HUT50871A - Process for producing recombinant plasmid dna encoding human fibroblast-beta 1-interferon and escherichia coli comprising same - Google Patents

Abstract

A recombinant plasmid DNA pPR-IFN beta 1-13, coding for synthesis of human beta 1-interferon has a size of 3.9 k.b.p. and consists of the following elements: BamHI-BglII, a fragment of a size of 3.4 k.b.p. of vector plasmid obtained on the basis of pPR40 and pML24, EcoRI-Sau3A, a fragment of plasmid pIN beta -trp 7 of a size of 510 b.p. deposited on 12.01.87 under No. 1825 at the culture collection of the USSR Research Institute for Antibiotics. A method of construction of the above plasmid DNA comprises the preliminary construction of vector plasmid pPR124B, then the construction of intermediary plasmid pPR-IFN beta 1-123 with the subsequent construction of plasmid pPR-IFN beta 1-13. A strain of bacteria Escherichia coli VNIIGENETIKA VL 903 (pPR-IFN beta 1-13) as producer of human beta 1-interferon deposited on 12.01.87 under No. 1825 at the culture collection of the USSR Research Institute for Antibiotics.

Description

The present invention relates to a novel recombinant plasmid DNA encoding human fibroblast-pi interferon, pPR-IFN p13, and to Escherichia coli VNIIGENETIKA VL 903 (pPR-IFN pi-13) producing human fibroblast-pi interferon.

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Interferons are proteins produced by specialized human cells in response to viral infections or to various inducers. Currently available experimental data indicate that interferons exhibit antiviral, anti-infectious and immunomodulatory properties when treated with interferon in certain cells, tissues and organs. In terms of its general application and importance, the interferon system for the body is comparable to the immune system. In terms of biological, chemical, and antigenic properties and production cells, human interferons are divided into three groups: α-leukocyte, β-fibroblast, and immune interferons, which are mainly produced by leukocyte, fibroblast, and T-lymphocyte cells.

The therapeutic applications of interferons have largely been identified. These include keratitis and dermatosis, and respiratory tract infections caused by various viruses (Grippe, adenoviruses), Hepatitis B and other viruses.

However, the mechanism of action, structural and functional properties and clinical potential of interferons have not yet been sufficiently investigated. This is due to difficulties in the preparation of pure interferons in a preparative manner by conventional methods due to the use of induced human cell cultures as a protein source. In addition, interferons synthesized in this way are usually complex mixtures of different subtypes and forms, the components of which differ in structural and functional characteristics. This makes it very difficult to investigate the properties of each interferon. As a result, the microbiological post-synthesis of individual interferons is becoming increasingly widespread.

Several methods are known for the production of human o, β and β interferons by bacteria, in particular various strains of Escherichia coli, Bacillus subtilis, Pseudomonas, which contain recombinant plasmid DNA which promotes expression of heterologous genes in a new genetic environment. In the case of human fibroblast interferon (IFN βΐ), the expression of the IFN βΐ gene is

The E. coli genes and colifags (P ^) encode the mature interferon in E. coli cells.

UV 5 trp) transcription (tuf B, rec A) and operons (lac and translational signaling). Mature interferon βΙ (molecular weight ca. 19000 Dalton) resulting from microbiological synthesis was deficient in a carbohydrate component and in the methionine moiety characterized by the presence of a formylmethionine N-terminal group, however, this difference does not lead to a change in the biological activity of the protein product of the IFN βΐ gene, similar to the native glycoprotein produced in fibroblast cells. molecular mechanism, even for medical purposes.

Recombinant plasmid DNAs encoding the synthesis of human fibroblast-pi interferon are known, such as pC1857 and pPLc245HFIF25 and E. coli SG40444,

5 containing the said plasmids (Remaut E, Staussens P., Fiers G, 1983, Nucl. Acida Res. 11, 4677-4688).

In the recombinant pPLc245HFIF25 plasmid DNA composition, the transcription of the IFNβ1 gene is regulated by the P1GG regulatory element of the lambda bacteriophage, and the translation of the protein product of the gene into the hybrid fragment of the ribosome binding site is based on the SD2 solved. The maximum yield of human? -Interferon that can be achieved by culturing said strain is 4% of the total protein content of E. coli containing the plasmid.

This strain is characterized in that it requires a plasmid pC1857, which is located on the same temperature-sensitive regulator gene of clts857, for the control of the expression of the IFN pi gene by the P1 promoter. This leads to a potential instability of the recombinant plasmid due to recA-dependent recombination between pC1857 and pPLc245HFIF25 in large-scale commercial culture, due to recombination beyond homology domains. In addition, the use of the relatively short SD sequence of the MS2 phage replicase gene to construct the hybridosome of the ribosome binding site does not fully allow for the potential performance of the E. coli translation apparatus, since the introduction of translocation of fibroblast interferon with non-ribosomes effective enough. However, the lack of transcriptional (? -Independent terminators) at the 3 '' end of the IFN p-1 gene of pPLc24HFIF25 is untranslated in the copy. In addition, a significant reduction in the production of numbers results in the loss of the recombinant plasmid in the case of depression of the highly efficient β 1 promoter of the lambda phage, which initiates transcription of the IFNβ1 gene.

This does not allow large-scale synthesis of the IFNβ gene product and may lead to further reduction of the pi-interferon content of the biomass when industrially grown, which makes it difficult to isolate the pure protein and reduces the yield of the final product.

The recombinant plasmid pPR-IFNpI-13 DNA produced by the method of the invention and the strain Escherichia coli VNIIGENETIKA VL 903 (pPR-IFNpI-13) containing it are novel, not described in the literature.

There was a need for a novel recombinant plasmid DNA encoding the synthesis of human fibroblast-pi interferon and for a high-throughput strain containing it that permits its production on pi-interferon in high yield.

Recombinant plasmid DNA, pPR-IFNβ-13, which encodes for the synthesis of human fibroblast pi interferon, was prepared and contains 3.9 thousand base pairs (kb) and consists of the vector pPR 124B. a 3.4 kb BamHI-BglII fragment of plasmid plasmid, constructed from the 510 bp fragment of plasmid pINp-trp7, pPR40 and pML24-EcoRI-Sau3A, which is characterized as follows:

contains a section that replication in. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · |

- Ί provides and regulates annak, the ColEI replicon, the lambda phage clts 857 repressor gene, the ampicillin resistance gene (Ap), the tdem of the ς-independent terminators of fd phage transcription, the PpOO regulatory domain and the SD cro gene which encodes a methionine codon on a mature human fibroblast interferon.

The coding region of the pi-interferon coding region and the cogene regulatory region encoding the transcription terminator of the fd phage is formed by forming a hybrid region for ribosome binding upstream of the IFN p1 gene, having the nucleotide sequence:

5 '-... TAAGGAGGTTGGARG ...- 3' where TAAGGAGGT is the SD sequence of the lambda phage cro gene, ATG is the methionine codon of the fibroblast interferon and is located immediately after the terminator codon of the IFN p1 gene tandem of φ-independent terminators of phage transcription:

- contains unique clal-restrictionase recognition sites, with clal coordinates indicating start of reading (0), PvuII (~ 1110), BglII (1480), AccI (^ 1870), Pvul (^ 3360),

- 12.01.1987, 1825, deposited in the Microorganism and Culture Collection of the Antibiotic Research Institute.

The recombinant plasmid pPR-IFNβ-13 produced by the method of the invention results in the expression of the IFN pi gene in the regulatory region of the lambda bacteriophage P ^ 0p.

• · · ····· ··· ··· ♦ · ··· ·

- With 8 controls.

The clts857 gene, which regulates transcriptional introduction of the early promoter of the lambda bacteriophage, and the IFN pi gene, which is regulated by the Pp promoter, are located in the same recombinant plasmid, pPR-IFN pi-13. This circumstance allows to eliminate potential instability in the cultivation of a strain containing a recombinant DNA molecule, since in the proposed construct no recA-dependent recombination occurs in the production strain in the absence of additional plasmid.

The method of constructing the recombinant plasmid pPR-IFN pi-13 according to the invention consists in cleaving the plasmid pPR124B made of plasmids pPR40 and PML24 with restriction endonuclease EcoRI and BglII, the largest fragment of the vector EcoRI-Sau 3A obtained being the pINp- trp7, which contains the coding region on the mature pi interferon. The resulting recombinant plasmid pPR-IFN pi-123 is inserted into the SD sequence of the cogene and the first methionine codon on the pi interferon is hydrolyzed with BamHI, a portion of the nucleotides being digested with E. coli DNA. polymerase I exonuclease, which are present in the reaction mixture in incomplete proportions of nucleoside triphosphate. The DNA was digested with EcoRI restriction enzyme and treated with S1 endonuclease to remove single-stranded DNA fragments, then the double-stranded ends were repaired with the Klenow fragment of E. coli DNA polymerase and the resulting linear DNA molecules were cleaved with T4 phage DNA ligase. cyclized. The resulting composition was E.coli C600

- 9 cells were transformed on culture medium containing ampicillin at 28 ° C by removing the transformants, then selecting the clones which proliferate at 42 ° C, and then selecting the recombinant plasmid pPR-IFN pi-13 from the selected clones.

By constructing the recombinant plasmid pPR-IFNpI-13, which encodes a particularly long SD sequence of the lambda bacteriophage cro gene, which is encoded by the mature human interferon portion of the coliforms, known as E. coli matrix RNA, A hybrid region of the pi gene ribosome binding site is obtained which has the optimal primary and spatial structure of the 5 'end of the matrix RNA. In contrast to known methods, the method of the present invention provides for the presence of a tandem of the q -dependent transcriptional terminator in the 3 'untranslated region of the IFNβ1 gene in plasmid pPR-IFNβ1, which results in high levels of plasmid DNA replication and fibroblast interferon gene. interference with its efficient transcription in the case of depression of the? 1 promoter.

In addition to the foregoing, the present invention also relates to the production of an Escherichia coli strain, the Escherichia coli strain VNIIGENETIKA VL 903 (pPR-IFN pi-13), which produces human interferon and which contains the recombinant plasmid DNA pPR-IFNpi-13 according to the invention. , produced genetically by introduction of recombinant plasmid DNA, pPR-IFNβ-13 into Escherichia coli, and on December 12, 1987, in the Microorganism and Culture Collection of the Antibiotic Research Institute. «'·. · · · · · · · · · · · · · · · ·

- 10 have been deposited.

The strain of the invention allows human pi interferon to be performed under large-scale industrial conditions

Produced by Q culture with a yield of 2.10 units / l or 5-10% of the total cellular protein

The most preferred embodiment of the process of the invention

The preparation of the recombinant plasmid DNA pPR-IFNβ1-13 according to the invention is carried out in several steps.

As a first step, a plasmid vector pPR124B is constructed.

To do this, a deletion of the Bal31 exonuclease was made in the plasmid pPR40 (Molecular Biology, 1987, Vol. 21, Vol. 5, Moscow, pp. 1309-1321) at the recognition site of the BglII restriction enzyme.

Thus, plasmid pPR100, in which a

The BamHI recognition site is located immediately downstream of the lambda phage ribosome binding site, resulting in the following sequence in the AUG codon to be initiated:

N GATCC,

Bam

Subsequently, the smaller PstI-BamHI fragment of plasmid pPR101 is ligated with the larger PstI-BamHI fragment of plasmid pML24 by conventional genetic engineering techniques to form plasmid pPR124, which is inserted into a BglII restriction site recognition site using an oligonucleotide linker. This gives plasmid pPR124B.

»· · · · ·•••••••••••

The intermediate plasmid pPR-IFNβ123 was then constructed.

Plasmid pPR124B was cleaved with restriction endonucleases EcoRI and BglII, and the larger of the resulting vector molecule fragments was ligated with the EcoRI-Sau3A fragment, which contains the coding portion of the mature pi-interferon from plasmid pINp-trp7. Thus, the recombinant intermediate plasmid pPR-IFN? 51 -123 was obtained.

Next, the third step involves constructing plasmid pPR-IFNβ-13. This involves approximating the SD sequence of the cogene in plasmid ρPR-1FNJ51 - 123 and the first codon of pi-interferin, to which the plasmid DNA is hydrolyzed with the restriction enzyme BamHI, and some of the nucleotides are removed by E. coli DNA polymerase I endonuclease, the DNA was digested with EcoRI restriction enzyme S1 endonuclease and then treated with the Klenow fragment of E. coli DNA polymerase I to repair the double chain, and the resulting linear DNA molecules were cyclized with T4 phage DNA ligase.

The resulting preparation is used to transform E. coli C600 cells. Transformants were grown on ampicillin-containing medium at 28 ° C and then selected at 42 ° C at a reduced growth rate. Clones thus selected express plasmid pPR-IFNβ-13 DNA.

The bacterial strain of the present invention, Escherichia coli VNIIGENE1IKA VL 903 (pPR-IFN pi-13), was prepared by transforming the host strain with the recombinant plasmid pPR-IFN? lose12 • ·· ···· · · · ···································································································································································································· · ·. ' ... ·.:. ··; · Determination of the activity of fibroblasts on pi-interferon after depression of the Pp promoter in extract of transformant cells, which is achieved by culturing the strain for 1-2 hours at 42 ° C.

The host strain is E.coli C600 or other E.coli

A K12 derivative may be used.

The characteristics of Escherichia coli VNIIGENETIKA VL 903 (pPR-IFN pi-13) are as follows.

Morphological characteristics

Cells are straight, rod-shaped cells (1.2-1.6) x (2.0-6.0) / µm, which are poorly mobile, gram-negative, spore-free, forming filaments.

Breeding characteristics

Cells grow well in liquid and solid or semi-solid, simple synthetic, semi-synthetic or complex media. Grown on Hottinger-bouillon and L-bouillon agar, they form mucus-like, round, poorly matte colonies. When grown in M9 liquid medium with L-bouillon type or casaminic acid, they form a homogeneous suspension.

Physiological-biochemical characteristics

Cells can grow at temperatures between 5 and 40 ° C (optimum around 35 ° C), pH 6.7 to 7.5. Carbon sources include amino acids and synhydrates (e.g., sucrose). As the nitrogen source, inorganic salts include ammonium salts or organic compounds such as peptone, tryptone, yeast extract and amino acids.

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- 13 Antibiotic resistance

The strain is resistant to ampicillin up to 100 mg / l when grown on liquid and agar media.

Stability of the plasmid

When cells are stored on agar medium (up to one month), serial passage (at least 6 months) and cultured in liquid antibiotic medium by submersion, no loss is observed and the plasmid is not transformed.

The Escherichia coli strain VNIIGENETIKA VL 903 (pPR-IFN pl-13) produced is highly efficient in the production of human fibroblast-III interferon and can be used commercially for the production of pi interferon.

The present invention is illustrated by the following examples, which illustrate the construction of the plasmid of the invention, the construction of the strain, and the culture thereof, as well as the figures.

Figure 1 shows a genetic map of the recombinant plasmid pPR-IFNβ1-13.

Figure 2 is a schematic diagram of the construction of the plasmid pPR-IFNβ1-13 according to the invention.

Example 1

The recombinant plasmid pPR-IFNβ-13 (Figure 1) was constructed in several steps. The first step is the construction of the plasmid pPR124B vector.

To do this, plasmid pPR124 was first constructed as follows. 3 .mu.g of plasmid pPR40 DNA • ···· ·· 60 microliters of buffer solution "·· · · · ·,:;

• ··· ··· «« * _β * * · · ··· ♦ · ♦ * ·· ··· · ««,

- 14 bar (restriction buffer 1: 10 mM Tris-HCl, pH 8.0, 6 mM MgCl 2, 6 mM 2-mercaptoethanol, 150 mM NaCl) was digested with BglII. . The DNA was precipitated with double volume ethanol. The precipitate was dissolved in 20 µl of water. DNA for 3 minutes at 30 ° C in 30 µl buffer (3 mM NaCl, 60 mM CaCl 2, 60 mM MgCl 2, 100 mM Tris-HCl, pH 8.0). 5 mM ethylene diamine tetraacetic acid) treated with Bal31 exonuclease. The DNA was precipitated with double volume ethanol. The precipitate was dissolved in 10 µl of water. The 30 µl sample prepared with restriction buffer 1 is treated with BamHI. The single-stranded 3'-end was supplemented with a Klenow fragment of E. coli DNA polymerase I in a 20 µl sample of 10 mM Tris-HCl, pH 8.0, 10 mM MgCl ^. yug DNA preparation, 30-30 µM / l of each DNA and 5 units of enzyme. The DNA was precipitated from the reaction mixture with ethanol and dissolved in 100 µl of water. The linearized plasmid DNA was pooled at 16 ° C for 12 hours in phage T4 DNA ligase in ligated buffer (60 mM Tris-HCl, pH 7.6, 10 mM MgCl 2, 10 mM). 1-2-mercaptoethanol, 0.4 mmol / l adenosine triphosphate) containing 1 µg of DNA. The resulting mixture is used to transform E. coli C600 cells.

Transformation efficiency ranges from 5.10 to ⁶ colonies per liter of native plasmid pPR40. Clones that are resistant to ampicillin (100 µg / L) are selected, in which plasmid DNA is expressed by modified Bimbolim and Doly methods and is subjected to restriction analysis.

··· · * ··. As a result, plasmid pPR100, which contains a unique BamHI site, is obtained. The plasmid pPR124 was then constructed.

For this, 2 µg of plasmid pML24 DNA was cleaved with 40 µl of restriction enzyme BamHI and PstI in restriction buffer 1. These were combined at 0 ° C with the fragments obtained by digestion of plasmid pPR 100 with BamHI and PstI using phage T4 DNA ligase. The resulting DNA preparation is used to transform E.coliCeOO cells, the transformants are selected on medium containing ampicillin and then 300 µg / L chloramphenicol by selection of Cm ^r clones at 42 ° C. Plasmid DNA was isolated from the clones thus selected and analyzed by restriction analysis. As a result, plasmid pPR124 is obtained, which contains a unique BamHI site. 2 µg of plasmid pPR124 DNA were then added in 70 µl restriction buffer 1 (10 mM Tris-HCl, pH 7.9, 6 mM MgCl 2, 6 mM 2-mercaptoethanol and 150 mM). (NaCl) was cleaved with XbaI. The DNA was precipitated with double volume ethanol. The precipitate was dissolved in 15 µl of water. Restoration of the 3 'end of single-stranded DNA was performed using a Klenow fragment of E. coli DNA polymerase in 20 µl of 10 mM Tris-HCl, pH 8.0.

mM DNA, 2 µg DNA, 30-30 µM DNA, and 5 units enzyme composition.

The DNA was precipitated from the reaction mixture with ethanol and dissolved in 100 µl of water. Ligation of the linearized plasmid DNA using the BglII linker was performed at 16 ° C for 12 hours.

Using phage T4 DNA ligase ligated buffer (60 mmol / 1 Tris-HCl, pH 7.6, 10 mmol / 1 _MgCl2, 10 mmol / 1 2-mercaptoethanol, 0.4 mmol / 1 ATP , 2µg plasmid DNA, 0.5µg linker).

After ligation, the DNA is precipitated from the reaction mixture with ethanol, dissolved, and hydrolyzed with BglII in 40 µl solution containing restriction buffer 2 (60 mM Tris-HCl, pH 7.6, 6 mM). MgCl ₂ , 6 mM 2-mercaptoethanol, 50 mM NaCl). The DNA is again precipitated with ethanol, dissolved and cyclized by treatment with 1 µg of plasmid DNA preparation in 240 µl of ligation buffer with phage T4 DNA ligase. The resulting mixture is used to transform E. coli C600 cells. Transformation efficiency is 5.10 µg per µg native plasmid pPR124. Clones that are resistant to ampicillin (100 µg / L) are selected, and plasmid DNA is selected from them by the modified Bimbolim and Doly method and subjected to restriction analysis. As a result, plasmid pPR124B is obtained which, unlike the parent vector pPR124, contains a unique BglII site instead of the XbaI recognition site (Figure 2).

Step Two: Construction of pPR-IFNβ1-123 Plasmid

The vector encoding the human fibroblast interferon sequence from plasmid pIN {5-trp7 is integrated into pPR124B. To do this, 10 µg of pINp-trp7 DNA, together with restriction enzymes EcoRI and Sau3A, were treated in 100 µl sample containing restriction buffer 2. The preparation was prepared with 1.1% readily. ········································································•

- Transfer onto 17 melting agar vapor gels and electrophoreses in tris-acetate buffer system. A band of the gel containing a 510 bp DNA fragment was excised and the DNA eluted from the gel. The DNA fragment thus produced (about 1 µg) was integrated into plasmid pPR124B. For this, 1 µg of pPR124B DNA with EcoRI and BglII was added at 20 µl,

2) in a sample containing restriction buffer. The preparation was subjected to phenol deprotection, the DNA was precipitated with ethanol and dissolved in 10 µl of water. 0.5 µg of cleaved pPR124B DNA was combined with 1 µg of the excised plasmid pINptrp7 fragment and treated with phage T4 DNA ligase in a 30 µl sample containing ligation buffer. The resulting DNA preparation is used to transform E.coli C600 cells, the transformants are selected on culture medium containing ampicillin and then cultured at 42 ° C in medium containing 200 µg / l chloramphenicol. Plasmid DNA was isolated from the clones thus selected and subjected to restriction analysis. In the resulting plasmid, the EcoRI-BglII framework, which encodes the C-terminal region of chloramphenicol ethyl1-transferase in the pPR124B vector, is replaced by its mature human p1 interferon coding sequence. This plasmid is designated pPR-IFNβ1-123 and used in the next step of construction.

Step Three: Construction of Plasmid ρPR-1FN? 51 -13 30 µg of plasmid pPR-IFNpI-123 were digested with restriction enzyme BamHI in 150 µl restriction buffer 1, the DNA was deprotected with phenol and precipitated with ethanol. The precipitate was dissolved in 40 µl of water. The DNA has limited deg • · · * • ♦ ♦ ♦ · · · ♦ ♦ ♦ ♦ «« «« · ·

- For 18 radiations with E. coli DNA polymerase I exonuclease in the 3 'to> 5' direction, the resulting DNA preparation was incubated in a 150 µl sample containing 50 mM Tris-HCl (pH = 8.0), 10 mM ethylenediaminetetraacetic acid, 5 mM MgCl 2, 100 μM deoxyadenosine triphosphate (then deoxy guanosine phosphate) and 20 units DNA polymerase. The reaction was stopped, the DNA was precipitated, and the precipitate was dissolved in 40 µl of water. 10 µg of the prepared preparation was treated with EcoRI restriction enzyme in 50 µl sample containing restriction buffer 2, phenol deprotection followed by ethanol precipitation and the precipitate was dissolved in 30 µl water.

The single-stranded sections of the prepared DNA preparation were removed using a S1 endonuclease in a 100 µl sample containing 30 mM sodium acetate (pH 4.4),

Contains 4.5 mM ZnSO4, 250 mM NaCl, 10 µg DNA and 200 units S1 endonuclease. The reaction was carried out at 20 ° C, then the mixture was subjected to phenol treatment and the nucleic acid was precipitated with ethanol. The precipitate was dissolved in 20 µl of water. 4 µg of the thus prepared DNA were treated with the E. coli DNA polymerase I Klenow fragment under the conditions described above to fill any single-stranded ends that may be present. The reaction is stopped by deprotection, the nucleic acid is precipitated with ethanol and dissolved in 20 µl of water.

The ligation of the double-stranded linear DNA molecules was carried out at 16 ° C in a volume of 50 µl of 60 mM Tris-HCl, pH 7.6, 10 mM MgCl 2, 10 mM dithiothreitol. , 4 mmol / l adenosine triphosphate, 8 wt% ···················································•

19 polyethylene glycol 6000, 4 µg DNA and 100 units of T4 phage DNA ligase. After completion of the reaction, the mixture is diluted four-fold, the nucleic acids are precipitated with ethanol, 2 µl of DNA dissolved in water are treated with 30 µl of restriction enzyme BglII, the DNA is precipitated from the reaction mixture with ethanol, dissolved in 200 µl of ligation buffer. in ligation with phage T4 DNA ligase. The resulting DNA preparation was used to transform E.coliC600 cells as described above, and the transformants were selected by culturing on ampicillin-containing medium for 24 hours at 28 ° C. From the resulting transformants, variants are selected which show a reduced growth rate at 42 ° C. From these clones, plasmid DNA was selected as described above and subjected to restriction analysis.

In the thus produced plasmid pPR-IFNβ-13, 30 µg of the appropriate DNA was prepared by digestion with 30 µl of restriction enzyme HindIII in 100 µl of restriction buffer 2, prepared on a 4-12% polyacrylamide gel gradient to generate the primary structure of the hybrid region of the ribosome binding site of the IFNβ gene. and subjected to electrophoresis in a tris-acetate buffer system. The 190 bp DNA fragments were eluted by the Maxam-Gilbert method and ligated to the linearized plasmid pPR124 in a similar manner to the BglII linker described above using phage T4 DNA ligase. The DNA is precipitated from the reaction mixture with ethanol, the precipitate is dissolved in 20 µl of water and 30 µl of BamHI

Cleavage buffer 1, DNA with phenol · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

It is deprotected, precipitated again with ethanol and dissolved in water.

Molecular cloning of the DNA fragment obtained by BamHI digestion and the DNA replicative form of bacteriophage M13mpl0, selection of recombinant phages on indicator medium containing 5'-bromo-4-chloro-3'-indolyl-β-galactoside, expression of single-stranded phage DNA the primary structure of cloned fragments is determined by limited matrix copies (F.Senger's method) by standard methods.

Nucleotide sequence of the hybrid sequence of the ribosome binding site in pPR-IFNp1-13 according to the Senger method:

5 '-... TAAGGAGGTTGGATG ...- 3', wherein TAAGGAGGT is the SD sequence of the phage gene of the lambda phage, and ATG is the methionine code for interferon p-1.

Example 2

The human fibroblast-β1 interferon-producing strain VNIIGENETIKA VL 903 (pPR-IFN pi-13) is produced as follows.

Plasmid pPR-IFNβ-13 encoding human fibroblast interferon was transformed into E. coli strain VNIIGENIKA VL 903 as in Example 1. Said strain was deposited under VKPM B-3546 at the Federal Institute for Genetics Research, Collection of Industrial Microorganisms.

Transformation efficiency of E. coli VNIIGENETIKA VL 903 cells is about 10 µg clone per 1 µg of native pPR-IFNβ-13 plasmid DNA. Transformants are obtained from medium containing 100 µg / l of ampicillin and the medium containing 100 µg / l of ampicillin.

- 21 cultures for 24 hours at 28 ° C. Plasmid DNA was isolated from the selected clones and their identity to pPR-IFNβ1-13 DNA preparations was confirmed by restriction analysis. This gives E. coli VNIIGENETIKA VL 903 (pPR-IFN P1-13).

To determine the productivity of E. coli VNIIGENETIKA VL 903 (pPR-IFN {J-13), plasmid containing cells were cultured at 28 ° C on standard Hottinger's medium inclined agar containing 50 µg / ml ampicillin for 14 hours. The biomass obtained on the inclined surface is used as an inoculum. Cells were transferred to 750 ml Erlenmeyer flasks containing 100 ml Hottinger's medium containing 100 µg / ml ampicillin and cultured at 28 ° C at 240 rpm for 6 hours. The optical density of the inoculum is 1.5-2.5 units.

The fermentation is carried out in a fermentor equipped with a pH, temperature, agitation and aeration control system. Fermentation is performed on Hottinger's medium containing 100 µg / ml ampicillin and 10 µg / l glucose. The inoculum is added in an amount of 5-10% by weight. The culture was carried out at pH 6.6-6.8 and maintained by the addition of ammonium hydroxide solution. The first part of the fermentation was continued at 28 ° C to 3.5 optical density at 550 nm, then thermoinduced by raising the temperature to 42-45 ° C for 5 minutes and then continued at the same temperature for 2 hours. .

At the end of the procedure, interferon cells were determined from 1 ml of culture broth to determine activity.

- precipitated by centrifugation at 22, resuspended in 1 ml of 0.02 M phosphate buffer solution containing 1% sodium dodecyl sulphate and 1% 2-mercaptoethanol at pH 7.2, and Heat at 100 ° C for 2-4 minutes. The precipitate was then centrifuged and the supernatant interferon content was determined by standard methods, either by protection against the cytopathological effect of vesicular stomatitis virus in human diploid fibroblast cells or by immuno-enzyme analysis. [3-interferon preparation (Torey, Japan), which is titrated to leukocyte interferon B 69/19 (UK), is used as a standard.

According to different assays, the activity of human fibroblast interferon synthesized by E. coli VNIIGENETIKA VL 903 (pPR-IFN J51-13) cells,

2.10 above international units.

Determination of synthesized fibroblast interferon relative to whole cell protein was performed by precipitation of 1 ml of culture broth by centrifugation, treatment as described above, separation of the preparation in 1.0% sodium dodecyl sulfate on 15% polyacrylamide gel and electrophoresis on R-250. NSK) Stained in Kumassi's solution by standard method. The quantitative protein content of the zones is determined by scanning an automatic laser densitometer. Identification of zones corresponding to mature fibroblast interferon (19kD) synthesized in cells by comparison with the electrophoretic mobility of the marker proteins and nitrocellulose-separated protein fractions. · * · ···

immunoprecipitated on a strainer and processed in a solution containing monoclonal mouse antibody and anti-mouse rabbit antibody to interferon-β, conjugated to horseradish peroxidase.

After proper staining, the interferon zones appear as a dark brown streak on the unpainted background of the nitrocellulose filter.

The protein content of the zone, which corresponds to interferon pi, is about 10% of the total cellular protein of E. coli VNIIGENETIKA VL 903 (pPR-IFN pi-13).

Industrial applicability

The recombinant pPR-IFN pi-13 plasmid DNA produced by the method of the invention, which encodes human fibroblast P1 interferon, can be used to produce cells that produce high levels of human P1 interferon.

Prepared by the process of the invention

Escherichia coli VNIIGENETIKA VL 903 (pPR-IFN pl-13) bacterial strain, which produces human interferon, can be used in the microbiological and pharmaceutical industry for the production of human pi interferon for use in medicine.

Claims (3)

CLAIMS 1. A recombinant plasmid DNA, pPR-IFNβ-13 encoding human fibroblast pi interferon, characterized in that it consists of 3.9 thousand base pairs and comprises the following elements: - the vector plasmid pPR124B, a 3.4 thousand bp BamHI-BglII fragment, made from plasmids pPR40 and pML23, 510 bp EcoRI-Sau3A fragment of plasmid pINp-trp 7, which has the following characteristics: - includes a section responsible for introducing and regulating replication, and Col El-rep lycda, the lambda phage inactivated 857 repressor gene, the Ap ampicillin resistance gene, the q-independent tandem of the fd phage transcription terminator, the P _R 0 _R regulator region of the lambda cro gene and its own methionine codon containing human mature IFNβ1 fibroblast interferon, - linking the regulatory region of the cRO gene, which encodes the p1 interferon coding region and the transcription terminator of the fd phage, by constructing a hybrid ribosome binding site region before the IFNβ1 gene having the nucleotide sequence: 5 '. ..TAAGGAGGTTGGATG. . .-3 ', in which TAAGGAGGT is located in the SD sequence of the lambda phage cro gene, and ATG is located after the methionine codon on the inter25 site and the tandem of the qd-independent terminator of the fd phage transcription, . - contains a unique Clal recognition site, where the coordinates represent the beginning of the reading (p), PvuII 1110), BglII 1480), Steel (^ 1870), Pvul (^ 3360), and on 01.01.1987, the Microorganism Research Institute and was deposited with his Breed Collection under number 1825. 2. A method for generating recombinant plasmid DNA pPR-IFNp13 according to claim 1, characterized in that the plasmid pPR124B produced from plasmids pPR40 and pML24 is cleaved with the restriction enzymes EcoRI and BglII, the larger fragments of the resulting vector EcoRI-Sau3A being pIN. Through the fragment of plasmid trn 7, which contains the coding region for the mature p1 interferon, the SD sequence of the cogene and the first (methionine) codon of the pi interferon are approximated in the resulting recombinant plasmid pPR-IFNp1 123. DNA is hydrolyzed with BamHI restriction enzyme, a portion of the nucleotides are removed using E. coli DNA polymerase I, which is recovered in the reaction mixture under incomplete conditions of the nucleoside triphosphate, and digested with EcoRI restriction enzyme. to remove single-stranded sections by treatment with S1 endonuclease followed by E. coli DNA polymerase I Klenow fra , the resulting linear DNA molecules were cyclized with phage T4 DNA ligase to produce the resulting E. coli C600 cell ···· ··. : ^β ········· · ··························································································································· · · and then separating the recombinant plasmid DNA pPR-IFNβ-13 from the selected clones. 3. Escherichia coli producing human 31 interferon VNIIGENETICS The bacterial strain VL 903 (pPR-IFN p13) containing the recombinant plasmid DNA pPR-IFN p13 according to claim 1 and genetically engineered into the bacterium Esprichia coli pPR.IFNpi-13. and was deposited at the Antibiotic Research Institute's Microorganism and Culture Collection on 18 January 1987 under No. 1825.