NL8100719A - MICROBIOLOGICALLY PREPARED POLYPEPTIDE WITH THE AMINO ACID ORDER OF HUMAN INTERFERON, DNA AND PLASMID, CODING FOR THIS ORDER, MICROORGANISMS CONTAINING THIS GENETIC INFORMATION AND METHOD FOR MAKING THEREOF. - Google Patents

Abstract

There is utilised recombinant DNA technology in the production of interferon, which provides a microbiologically produced polypeptide, all or part of which comprises the amino acid sequence of human interferon, especially fibroblast (???) interferon. There is also provided CDNA which codes for such a polypeptide; a plasmid, especially plasmid pBR 322, which comprises the cDNA; and a microorganism, for example Escherichia coli, especially E. coli K12 or E. coli X1776, which comprises the genetic information required for the production of such a polypeptide. The cDNA is preferably prepared using mRNA obtained from human fibroblasts.

Description

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Betr.Y Microbiologically prepared polypeptide having the amino acid sequence of human interferon, DM and pioms encoding this sequence, microorganisms containing this genetic information and method of making it.

It has been known for more than 20 years that interferon prevents virus production. In recent years, studies have also shown that interferon also inhibits cell division and regulates many immune system responses. All these results underscore the great therapeutic significance of interferon.

The amounts of interferon that have hitherto been useful for scientific and therapeutic studies have been recovered from fibroblast and lymphocyte cultures. However, both methods have failed to produce large amounts of interferon, or sufficient amounts of individual problems. This is too; at the same time the cause is that the knowledge in the interferon field is advancing very slowly and that only very few of the urgently desired studies in the cancer field could be performed. It is therefore necessary to break new ground in the production of interferon.

Such a path is now pointed out by the present invention. This involves the isolation of nucleotide sequences containing the genetic code for interferon, the synthesis of DM with this specific nucleotide sequence, and the transfer of this DM into a host microorganism in which the replication and expression of this DM takes place.

The invention particularly relates to the formation of genes for fibroblast interferon, the transfer of these genes into a microbe host, replication of host and gene expression of the gene by the host. This produces interferon protein with the biological and immunological properties characteristic of this protein.

To achieve the present objectives, one can proceed in the following manner, with the path shown as pre-prepared for various usable states.

Human fibroblasts are stimulated to maximize production of interferon mEM in the presence of a protein synthesis inhibitor using polyinosinic acid / polycytidylic acid. The cells are denatured and the RM collected as a precipitate with a cesium chloride centrifuge. After reprecipitation, the ffiRNA is separated from this RNA via an oligo-dT cellulose column. The recovered mRNA is completed into an RNA-DNA double strand with the aid of the enzyme reverse transcriptase. After degradation of the RNA strand, the DNA strand (the complementary DNA = cDNA) is reverse transcriptase or the enzyme polymerase I completed into a DNA double stranded DNA (dsDNA).

'This double-stranded DNA must now be built into a piasmi. To this end, the extension of the 31-end of the DNA, e.g. with dCMP residues. The plasmid (e.g. pBR 322) is cut open with the restriction nuclease PstI and e.g. extended with dGMP residues. When the thus extended DNA is combined with an appropriately extended plasmid, base pairing between DNA and plasmid takes place. This circularized molecule is then transformed into microorganisms (especially bacteria, most preferably E. coli v such as E. coli K 12 or X 1776); the not yet closed bonds are covalently cross-linked by the enzymes of the ant organism. The transformed microorganisms are streaked on agar plates and selected for antibiotic resistances given by the selected plasmid and the restriction nuclease used.

Using immunological and biological tests, the. 20 clones, which form interferon-containing proteins, were selected. These clones are grown, the microorganism mass centrifuged off, extracted and examined for interferon content. The solution recovered by digestion of the interferon-producing clones contains interferon protein.

The present steps can be carried out in the following manner:

1. Method for isolating RNA

a) Collection of interferon-producing fibroblast cells.

Human neonatal foreskin fibroblasts were propagated in roller passes (0 10 cm, length 10 cm) at 37 ° C under 30 using a suitable culture medium (e.g., Eagle's medium MEM) (minimal essential medium) plus fetal calf serum). After reaching washed cell cultures from 25th to 35th cell duplication, the propagation medium is replaced with the interferon induction medium. This consists of Eagle's MEM, 8100719-3 to which appropriate amounts of the fibroblast interferon inducer polyinosinic acid: polycytidylic acid (10-100 ^ g / cm; in actual

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case 50 µg / emJ) and the protein synthesis inhibitor cycloheximide (10-100 3 µg / cm, in this case 50 µg / cm.) were added. After incubation of the cell cultures at 37 ° C for k-6 hours (in the present case 5 hours), the supernatant was poured off,

b) Extraction of 5M

The obtained cells were taken up in a denaturing medium M guanadinium thiocyanate, 1 M mercaptoethanol, 0.15 M sodium acetate with 10 pH 5.5) and homogenized with an ultra-turrax for 1 minute at 0 ° C. The hydrogenate was centrifuged at 20000 rpm for 15 minutes at + + C.

The supernatant was then applied to a solution (pad) of

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5 cnr 5.7 M CsCl, 10 mM tris-hydroxyaminamethane (tris), 10 mM 15 ethylene diamine tetraacetic acid (EDTA) with pH 7.6 layered and in a Beckman-SW 27 rotor 36 hours at 20 ° C and 22,000 rpm centrifuge !.

It has been found that, contrary to the brief in the literature, addition of CsCl to the lysate should be avoided in order to achieve the most complete separation of mRWA in the next reaction step. At the end of the centrifugation, the polyalliter tubes were frozen in liquid nitrogen and the bottom of the tubes containing the RWA precipitate were cut off.

The precipitate was taken up in a solution of 10 mM tris, 10 mM EDTA and 10% "sarcosyl" (W-lauryl-sarcosine sodium salt) with pH 7.6 and the suspension was centrifuged at 20,000 rpm for 20 minutes. The resulting precipitate was taken up again in the same buffer, heated at 6> 5 ° C for 5 minutes and centrifuged again. The combined supernatants were made 0.3 M of sodium acetate, mixed with the 2.5-fold volume of ethanol and stored at -20 ° C.

2. Extraction of mRITA

The RITA was separated from the ethanol solution (20,000 rpm, 30 minutes, -5 ° C) by centrifugation and dissolved in 0.5 M NaCl, 10 mM tris, pH 7.5. This solution was applied to an oligo-dT cellulose column 35 (Collaborative Research, type 3) equilibrated with the same buffer, and was distilled with 1 mM tris, pH 7.6 or distilled with 1 mM water eluted. The elution of the poly-A-containing RITA (mRNA) can be monitored by an extinction measurement at 260 nm. The mRNA thus obtained was endowed with sodium acetate to a final concentration of 0.3 M, the 2.5-fold volume of ethanol and the solution is stored at ^ 20 ° C.

3. Extraction of the cDKA

a} Single stranded cDNA recovery

The conversion of the mRNA into the corresponding DM (cDM) 10 takes place with the aid of the enzyme reverse transcriptase.

The incubation mixture contained: 50 mM tris, pH 8.3, 10 mM MgClg, 30 mM 2-mercaptoethanol, 0.5 mM to all commonly occurring deoxyribonucleoside triphosphates (the triphosphates of adenine, 3 guanine, cytosine and thymidine), 100 / ug / cm oligo-dT12_1g (from 15 Boehringer Mannheim) approx. 100 jag / cm? polyadenylated RNA and 800

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units / cm "1 reverse transcriptase (from Life Science Ine.,

St. Petersburg, USA). To monitor the reaction, a c (-place) 32 P-labeled deoxyribonucleoside triphosphate (specific activity 50 Ci / mmole) can be added to the mixture. The mixture is incubated at k2 ° C for 60 minutes, followed by a addition of 20 mM EDTA the reaction is stopped.

The solution is extracted with an equal volume of phenol saturated with water, the phenol is removed by shaking with ether and residues of ether are distilled off with nitrogen. Cannabis built-in deoxyribonucleoside triphosphates were separated by column chromatography over Sephadex G50 in 10mM tris, pH 9.0, 100mM NaCl, 1mM EDTA. The eluted cDM was precipitated by adding 0.3 M sodium acetate and 2.5 times the volume of ethanol. After centrifugation, the precipitate was taken up in 0.1 M NaOH and incubated for 30 minutes at 70 ° C or incubated in 0.3 M NaOH overnight at room temperature. The mixture was adjusted to pH 7.6 with 1 M HCl and 1 M tris, b) Generation of double stranded cDNA (ds DNA)

For the formation of the second DNA strand, the enzyme reverse transcriptase or the enzyme polymerase I can again be used.

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Formation of dsDKA with reverse transcriptase:

The incubation mixture (pH 8.3) "contained 50 mM tris, 10 mM MgClp, 30 mM 2-mercaptoethanol, 0.5 mM of the" above 4 deoxyribonucleoid triphosphates, 50 µg / cm cDHA and 800 units / cm reverse transcriptase. The reaction was carried out for 120 minutes "at if-2 ° C and quenched to a final concentration of 20 mM by the addition of EDTA. Then the phenol extraction and a gel permeation chromatography on Sephadex G50 took place as described above.

Formation of dsDNA with polymerase I:

The incubation mixture (pH 6.9) "contained 200 mM hepes, 0.5 mM of the" U deoxyribonucleoside triphosphates above, 10 mM MgCl 2, 30 mM 2-mercaptoethanol and TO mM KCl. The reaction can be monitored by adding 32 P-labeled deoxyribonucleotides (qp the & L sites) to the incubation mixture. The reaction was started with 800 units / cm 2 polymerase I and carried out at 15 ° C for 2 hours. The reaction was terminated by addition of 0.1% sodium dodecyl sulfate and 100 µg / 3 cm RHA. The solution was extracted as described above. The solution obtained after the extraction was applied to a Sephadex G50 column and chromatographed with 10 mM tris pH 9.0, 100 mM jtfaCl and 1 mM EDTA. The eluted DHA can be determined by its radioactivity and was precipitated by adding 0.3 M sodium acetate and 2.5-fold volume of ethanol. The ethanol precipitate was taken up in a solution (pH 1.5) from 300 mM EaCl, 30 mM sodium acetate and 3 mM ZnClp and mixed with 1500 units / cm 2 S1 nuclease (Boehringer Mannheim).

The reaction was stopped after 1 hour at 3T ° C by adding EDTA to a final concentration of 20 mM. Then it was extracted with phenol and ethanol precipitated both as described above.

c) Extraction of cDM with a single reactor process.

Alternatively, the dsDNA from the mBWA can also be extracted using a single act or process. The reaction of the reverse transcriptase is then carried out as described, but still 10U mM SCI is added to the incubation mixture. At the end of the reaction, the 8100719 5-6 sample was heated at 100 ° C for U minutes and the precipitate formed was centrifuged off. An equal volume of 0.1 M hepes buffer (pH 6.9) was added to the supernatant, the above k deoxyribonucleotides being present in the buffer 0.5 mM.

By adding 800 units / cm polymerase I, the reaction was run as described at 15 ° C and stopped by adding sodium dodecyl sulfate and RNA. Then proceed as under 3b) "Formation of dsDNA with polymerase I". B. Extension of dd 3 'ends of cDNA with dCTP (deoxycytosine triphosphate).

Incorporation into the plasmid requires extension of the 3-terminus of the DNA with one of the above k deoxynucleotides. Furthermore, the 3 'ends of the cut open plasmid were extended with complementary deoxynucleotide. When DNA is combined with plasmid, a base pairing takes place between DNA and plasmid.

This recirculated molecule can be used for a transformation of bacteria; the not yet closed bonds are covalently cross-linked in bacteria by an enzyme system.

E.g. the 3 'ends of cDNA can be extended with dCMP residues (deoxycyto-sin monophosphate residues) and the plasmid with dGMP residues (deoxyguanin dinemonophosphate residues). When these deoxynucleotides are used in the manner described and when the plasmid is opened by the restriction enzyme Pst I, a Pst I cleavage site is newly formed at each insertion site after the foreign DNA has been incorporated, so that after insertion of the plasmid the inserted DNA is can easily be cut out again with the restriction enzyme Pst I.

The precipitate after the alcohol addition after degradation with $ 1 nuclease was dissolved in an aqueous solution of pH 6.7 of 30 mM tris, 1 mM CoClg, 1 ^ 0 mM potassium cacodylate, 150 mM dCTP, 100 µg autoclaved gelatin hydrolyzate and 0.1 M dithioerythritol dissolved. 32 P-labeled dCTP can be used to monitor the reaction. The reaction was started by adding terminal deoxynucleotidyl transferase and carried out at 37 ° C for 10 minutes. At the end of the reaction time, the sample was placed on ice and the number of the incorporated dCMP residues was determined by radioactivity measurement in trichloroacetic acid to precipitate. In the reaction, he should preferably have added about 10 # of the originally present nucleotides; if this is not the case, the reaction can be continued by adding further enzyme. If too many dCMP residues have been added, the chain formed can be shortened using the enzyme S1 nuclease.

5. Method for integrating DNA into a piasmid.

A suitable circular plasmid, e.g. pBR 322, was cut with a restriction donuclease that recognizes only an order on the plasmid. It is advantageous if this cleavage site lies behind a strong or inducible promoter and / or is located so that an antibiotic resistance is influenced.

The plasmid thus linearized is then extended at the 3-end with one of the four deoxynucleotides.

This is e.g. done as follows: 30 µg plasmid is mixed with 50 units of Pst I restriction donuclease in the presence of 50 mM ICa, 6 mM tris, 6 mM MgClg, 6 vmM 2-mercaptoethanol and 0.1 mg / cm gelatin for 37 min at 37 ° C and incubated a pH of 7 »5. Then it is extracted with phenol and ether as described and the cut-up plasmid is precipitated with ethanol in the presence of 0.3 M sodium acetate. The extension of the 3 'ends of the plasmid with dGTP (deoxyguanidine triphosphate) takes place analogously to the extension of cDHA with dCTP described above.

The extended piasmid-DKA is then mixed with the extended ds cDHA in a solution (pH 8.0) of 0.1 M HaCl, 10 mM tris and 1 mM EDTA, heated at 5 ° C for 2 minutes, at 2 ° C for 2 hours C incubated and then slowly cooled to room temperature. The hybrid DMA thus obtained is then used for a transformation of E. coli.

6. Cloning of hybrid plasmid into E. coli.

The bacteria, e.g. E. coli X 1776 5 were grown at 37 ° C in 50 cm3 of a conventional culture medium to an optical density of Agoo = 0.5 - 0.6, sedimented, washed once with 10 mM tris pH 7> 5 and then in 0 0 cm van of a solution of 75 mM CaClp, 5 mM MgCl 4 and 5 mM tris resuspended with pH 8.0 and incubated in ice for 20 minutes. The cells were then sedimented and in

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2 cm 75 mM CaClg, 5 mM MgCl2, 5 mM tris pH 8.0 resuspended.

0.2 ml of this bacterial suspension were then mixed with 0.1

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cm hybrid DM solution and incubated on ice for 5 minutes.

Then it was heated to b2 C for 90 seconds and then with 0.2 cm-3. 3 culture medium mixed. 50-75 mm of this suspension was then streaked on tetracycline and ampicillin-containing agar plates and selected for antibiotic resistance.

7 · Isolation of strains producing interferon-containing proteins.

a) Immunological evidence.

Clones were tested for the expression of interferon-containing proteins using a test system developed by Broome and Gilbert (PMS 75, 27k6, (1978)). Clones, which form interferon-containing proteins, are linked by radioactive antibodies to these proteins with a subsequent autorad! Also visible, because an X-ray film was blackened in these places. To this end, up to 50 clones per nitrocellulose film were grown at 37 ° C for 2 days. The filters were then placed on an agar block containing 1 mg / cm lysozyme; an interferon antibody-coated FVC film was applied to the bacterial colony, and then incubated at k ° C for 2-3 hours. The PVC film was then incubated in a solution of 132 iodine labeled interferon antibody for 15 hours at 1 ° C. The specific activity of the solution was about 1 x 1Cr cpm / cm. After culturing and drying the film, this was autoradiographed.

b) Biological demonstration.

The clones were grown at 50 ° C overnight from a conventional culture medium at 37 ° C. The bacteria were then sedimented, washed twice with cold 10 mM tris with pH 8.0, 30 mM ICaCl and 20% sucrose in 30 mM tris, 8.0.

Resuspended 1 mM EDTA. It was then shaken for 10 minutes at room temperature, sedimented, resuspended in ice-cold water and incubated in an ice bath for 10 minutes. The bacteria were then sedimented and the top 8f 00 7 1 9 8

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-9- self-assayed test for the content of interferon-containing protein in a virus inhibition test.

8. Interferon protein extraction.

The clones, which showed an interferon activity in the different experiments, were grown in the usual culture media, the bacteria were centrifuged off and extracted with aqueous buffers.

The extraction solution contained interferon protein.

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Claims (7)

1. Microbiologically derived polypeptide, containing all or part of the amino acid sequence of human interferon. 2. A microbiologically derived polypeptide according to claim 1, which contains all or part of the amino acid sequence of human fibroblast interferon. CDNA, encoding the amino acid sequence of human interferon according to claims 1-2, obtained with mRM from human fibroblast cells. 1+. Plasmid encoding the amino acid sequence of human interferon according to claims 1-2, obtained according to claim 3 and a plasmid, preferably plasmid pBR 322. 5. Microorganisms, especially E. coli, with the genetic information for a biosynthesis of the. polypeptide according to claims 1 or 2. 6. Method for forming the polypeptide according to claim 1 or 2, characterized in that the genetic information for the biosynthesis of the polypeptide with fibroblast mRM, the gene obtained in a manner known per se in micro-organisms, is obtained. and which selects and cultivates microorganisms in a manner known per se, which produce this polypeptide, 7. Method for forming cDM according to claim 3, characterized in that RNA is recovered from fibroblasts, the mENA is isolated therefrom and cDM is prepared in a manner known per se. Process for preparing the plasmid according to claim U, characterized in that a plasmid, preferably plasmid pBR 322, is combined with the cDM according to claim 3. Method for forming a micro-organism according to claim 5, characterized in that a micro-organism, preferably E_. coli with a plasmid according to claim 1. 10. Process for the recovery of R1A, characterized in that a cesium chloride-free lysate is applied layered to an aqueous concentrated cesium-chloride solution, centrifuged and the RNA is recovered. 8100719