SU1572419A3 - Method of obtaining recombinant plasmide dna prw11 of prw12 coding omega-interferon - Google Patents

Abstract

The invention relates to genetic engineering, in particular to the construction of recombinant plasmid DNA for the production of interferons. The P9 A2 or E79 E9 plasmid is digested with AVA II restriction enzyme, the resulting DNA insert is digested with SA and 3A, a 189 bp fragment is isolated, the PER 33 plasmid is digested with ESOR I and PVU II restriction enzymes, a 389 bp fragment is isolated. then digested with SAU 3A enzyme, a 108 bp fragment is isolated and ligated with a 189 bp fragment, the resulting DNA is treated with HIND III and ligated with HIND III lincrated ER 103 plasmid, the resulting recombinant DNA is used to transform E. COLI HB 101 strain, the resulting PRHW 10 plasmid is digested with BAM H 1, incubated in the presence of the Klenow fragment of DNA polymerase 1 and four deoxynucleoside triphosphates, the plasmid is treated with NCO I enzyme, a large NCO I - ALUI fragment of P9 A2 or E79E9 plasmid is isolated, which is obtained by treating the plasmid with AVA II followed by N CO I and ALU I hydrolysis, and the recombinant plasmid DNA is used to transform E. COBI HB 101.

Description

The invention relates to genetic engineering, in particular to the construction of expression plasmids for obtaining interlerons, and specifically to a method for constructing expression plasmids pRHWl 1 and pRFVl2 encoding new interlerons.

The method is carried out as follows.

Human B-lymphoma cell line, such as Namalma cells,

treated with a virus, such as the Sendai virus. The mRNA formed from the stimulated Namalwa cells is isolated and used as a template for cDNA synthesis. To increase the yield of interferon-specific sequences during cloning, the mRNA preparation is separated in sugars with a density gradient into individual mRNA molecules of different lengths.

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It is preferable to collect (accumulate) mRNA in the region around 12S (approximately 800 - 1000 mRNA bases). In this region, mRNA specific for sЈ - and |3 -interferons sediments. The mRNA of this region of the gradients is concentrated due to sedimentation and dissolution in water.

The cDNA library is constructed using known methods. mRNA is supplemented with oligo-dt. Then, cDNA is synthesized by adding four deoxynucleoside triphosphates (dATP, dGTP9, dCTP, dTTP) and the reverse transcriptase enzyme in a suitably buffered solution for 1 h at 4-5°C. The cDNA/mRNA hybrid is purified by extraction with chloroform and chromatography on a gel column, e.g. through Sephadex G50. RNA is hydrolyzed by treatment with alkali (0.3 mol NaOH at 50°C, 1 h), and the cDNA is precipitated with ethanol after neutralization with an acidic sodium acetate solution. After addition of deoxynucleoside triphosphates and R. coli DNA polymerase-1 to the appropriate solution, double-stranded (double) synthesis is carried out, wherein DNA serves simultaneously as a template and as a primer due to the formation of a hairpin-type structure at its 3'-end (6 h at 15°C). After extraction with phenol, chromatography on Sephadex G50 and precipitation with ethanol, DNA in a suitable solution is treated with specific endonuclease SЈ,. In this case, the hairpin structure is disintegrated, as well as the DNA not converted into a double strand. After extraction with chloroform and precipitation with ethanol, the double-stranded DNA (ds DNA) is separated by size in sugars with a density gradient. For the next cloning step, it is preferable to use only ds DNA of 600 bp or more in order to increase the probability of obtaining clones that contain the complete coding sequence of new interrons. ds DNA longer than 600 bp is concentrated from gradients by

precipitation with ethanol and dissolution in water.

In order to increase the resulting ds DNA molecules, they must first be introduced into the appropriate vector and then into the E. coli bacterium. The vector is the pBR plasmid.

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322. This plasmid consists of a replicon and two selection markers. They confer resistance to the host against the antibiotics ampicillin and tetracycline (Ap11, Tc1). The gene for β-lactamase (Ap11) contains a sequence for recognition of the restriction endonuclease PstI. pBR 322 can be cut with PstI. The protruding

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The 3'-ends are extended by the enzyme terminal deoxynucleotide transferase (TdT) by placing dGTP in an appropriately buffered solution. At the same time, the dsDNA is also extended at the 3'-ends by the enzyme TdT by using dCTP. The homopolymer ends of the plasmid and dsDNA are mixed in appropriate concentration ratios and under suitable conditions of temperature, salt and buffer conditions.

E. coli strain HB 101 (genotype F-, hsds 20 (r-B, m-B) rec A13, , - proA2, lacVI, galK2, rpsL120 (Smr), xyI-5, mtl-1, supE44, lambda-) are treated by growing in CaCl. Competent E. coli HB 101 are mixed with DNA and, after incubation at 0°C, transformed with the resulting plasmid DNA by thermal heating at 42 C for 2 min. Then, the transformed bacteria are placed on tetracycline-containing agar plates (10 μg per 1 ml). Only E. coli HB 101 that have accepted the vector or recombinant vector molecule (Tch) can grow on this agar. Recombinant vector-dc. DNA molecules transmit

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the host genotype Ap TC, since due to the introduction of ds DNA into the B-lactamase gene, the information for the b-lactamase is destroyed. The clones are transferred to agar plates containing 50 μg/ml ampicillin. Only about 3% grow, which means that 97% of the clones contain the insertion of the dsDNA molecule. Of these, 28,600 clones are transferred individually to the wells (recesses) of microtiter plates containing nutrient medium, 10 μg/ml tetracycline and glycerol. After the clones have grown in them, the plates are stored at -70 C (cDNA library).

After thawing, the clones are transferred to nitrocellulose filters. These filters are placed on a tetracycline-containing nutrient agar. After

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increase in bacterial colonies; bacterial DNA is fixed on the filter.

The insert of the clone pER33, which contains the gene for IFN-ed-2-Arg, preferably serves as a sample. This piece of DNA is radioactively labeled by nictitration, using DNA polymerase I, dATP, dGTP, dTTP and 2G-aTP. Nitrocellulose filters are first pretreated without the addition of radioactive sample under suitable non-stringent hybridization conditions and then hybridized for about 16 hours with the addition of radioactive sample. The filters are then also washed under non-stringent conditions. Due to the low stringency (precision) of hybridization and washing, not only interferon-β2-A-containing clones are included, but also other interferon-containing clones whose sequences may deviate significantly from the sequences of the previously known interferon c. After drying, the filters are exposed to X-ray film. Blackening, which is clearly above the background level, indicates the presence of a clone with interferon-specific sequences.

Since the radioactivity signals are of variable quality, positive clones suspected of being positively reacting clones are grown on a small scale. The plasmid DNA molecule is isolated, digested with the restriction probe nuclease Pst I and separated by size by electrophoresis on an agarose gel (agarose gel) by the Southern method and transferred to a nitrocellulose filter. The DNA from this filter is hybridized with a radioactive denatured sample containing the IFN gene. The plasmid IF7 (stored in DS M under the number 2362) containing the gene for interferon oi-2-arg serves as a positive control. The autoradiogram clearly shows that clone F76E9 and clone P9A2 contain a sequence that hybridizes under non-stringent conditions with the interferon gene ob-2-Arg. In order to further characterize the ds DNA insert of clone E76E9 and clone P9A2, rhazmids of these clones were prepared on a larger scale. The DNA was digested with various restriction enzymes

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endonucleases, such as Alul, |Sau 3A, Bg III, Hinf I, Pst I and iHae III. The resulting fragments are separated in an agarose gel. By comparing them with the corresponding size markers, such as the fragments formed by digesting pBR 322 with the restriction endonuclease Hinf I or HaeIII, the fragment sizes are determined. The sequence of these fragments is determined. It was concluded that in the case of the insertion of clones E76E9 and P9A2, we are dealing with previously unknown interferonogens, namely omega-interferons.

The omega interferon information is used to digest the cDNA target with restriction endonucleases. The resulting fragments are ligated into the ds DNA form of Ml3mp9 and sequenced using the Sanger dideoxy method. Single-stranded DNA of the recombinant phages is isolated. After coupling of the synthetic oligomer, double-stranded syntheses are carried out in four separate preparations using the large fragment of E. coli DNA polymerase 1 (Klenow fragment). In each of the four partial reactions, one of the four dideoxynucleoside triphosphates (ddATP; ddCTP; ddCTP; ddTTP) is added. This results in statistically distributed chain breaks at sites where the DNA matrix contains an additional base to the corresponding dideoxynucleoside triphosphate. In addition, radioactively labeled dATP is used. At the end of the synthesis reaction, the products are denatured and the single-stranded DNA fragments are separated by size in a denaturing polyacrylamide gel. The gel is then exposed to X-ray film. From the autoradiogram, the DNA sequence of the recombinant Ml 3 phage can be determined. The insertion sequence of the various recombinant phages is determined using a suitable computer program.

The isolated cDNA clone E76ED for omega(Glu)-interferon is 858 bp long and has a 3-untranslated region. The region that encodes mature omega(Glu)-interferon,

extends from nucleotide 9 to nucleotide 524. Isolated cDNA of clone P9A2 for omega (Glu)-interferon, 877 base pairs long, encoding mature omega-interferon, extends from nucleotide 8 to nucleotide 523. 3 - The untranslated region in the case of P9A2 extends to the poly-A segment (area).

h The DNA sequence encoding omega-interferon is completely contained in clones E76E9 and P9A2. They begin at the N-terminal end of the amino acids cysteine-aspartic acid - leucine. Both mature omega-interferons have a length of 172 amino acids, which clearly deviates from the lengths of both known interferons, for example 166 (respectively 165) amino acids in the case of ob-interferons. Both omega-interferons

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g-interferons have a potential N-glycosidation site at position 78 of the amino acid (asparagine-methionine-threonine).

Comparison of the DNA of clone E76E9 and clone P9A2 reveals a difference. The triplet encoding amino acid 111 in clone E76E9 is GAG and codes for glutamic acid, while this triplet in clone P9A2 is GGG and codes for glycine. The two omega interferon proteins therefore differ by one amino acid and are designated as omega (Glu) interferon (E67E9) and omega (Clu) interferon (P9A2).

- Comparison of both omega-interferons with the hitherto known human ob-interferon gives the following picture (see table).

Omega Characteristics

Protein length in amino acids 172 Potential N-glycosylation site at position 78

Interferon alpha-A has only 165 amino acids.

Interferon alpha-H has a potential site for N-glycosylation at position 75.

B. coli HB 101 with plasmid E76E9 and E„ coli 101 with plasmid P9A2 were deposited (3.7.1984) in the German Collection of Microorganisms (ISM Gottingen) under the numbers DSM 3003, respectively DSM 3004.

To prove that newly found clones produce interferon-like activity, for example, clone E76E9 is cultured, and

EcoRISau3A

gaattcacgct

GATCGCTAAAACATTGTGCAAAMGAGGGTTGACTTTGCCTTCGCGA

ImRNA-Start

ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116

RBS Hindlll

Alpha

166

The bacterial lysate after its growth is tested in the spot reduction test. However, the genes are expressed in

expression plasmid pER 103, so

how this vector contains all the regulatory elements that lead to a high rate of expression of the cloned genes. According to the invention

in the plasmid pBR 322 the shorter EcoRI/Bam HI fragment is replaced by a DNA sequence of the formula

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9 157241910

Cys Asp is obtained with restriction endonuclease TRT gt gt™ZOY Ava11 fiocjie chromatography and

1Ы ыи i IJ-N - omega - Сэн - purification of the obtained cDNA insert its

Sau3A, digested with restriction endonucleases N col and Alul twice and then isolated with chromI. Obtaining the individual DNA fragments necessary for this matography and electroelution. purposes. This fragment contains most of the soFTEYAGMRN

10 corresponding omega-interferonogen.

To obtain a fragment of the plasmid P9A2, which contains the gene for IFN-1, the omega (C1y)-interferon clone P9A2 has the following structure:

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His Gly Leu Leu Ser Arg Asn Thr Leu CNcol CAT GGC STA CTT AGC

AGG AAC ACC TTG 28

202530

Val Leu Leu His Gin Met Arg Arg lie Ser Pro Phe Leu Cys Leu

GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 73

354045

Lys Asp Arg Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly

AAG GAC AGA AGA GAG TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 118

505560

Ser Gin Leu Gin Lys Ala His Val Met Ser Val Leu His Glu Met

AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163

657075

Leu Gin Gin He Phe Ser Leu Phe His Thr Glu Arg Ser Ser Ala

CTG CAG CAG ATS TTC AGC CTC TTC dAC ASA GAG CGC TCC TCT GCT 208

808590

Ala Trp Asn Met Thr Leu Leu Asp Gin Leu His Thr Gly Leu His

GCC TGG AAC ATG ACC CTC CTA GAC CAA CTC CAC ACT GGA CTT CAT 253

95100105

Gin Gin Leu Gin His Leu Glu Thr Cys Leu Leu Gin Val Val Gly

CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA 298

PO115.120

Glu Gly Glu Ser Ala Gly Ala lie Ser Ser Pro Ala Leu Thr Leu

GAA GGA GAA TCT GCT GGG GCA ATT AGC AGC CCT GCA CTG ACC TTG 343

125130135

Ar Ar Tyr Phe Gin Gly He Arg Val Tyr Leu Lys Glu Lys Lys

AGG AGG TAG TTC CAG GGA ATC CGT GTC TAG CTG AAA GAG AAG AAA 388

140145150

Tyr Ser Asp Cys Ala Trp Glu Val Val Arg Met Glu He Met Lys

TAG AGC GAC TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA 433

155160165

Ser Leu Phe Leu Ser Thr Asn Met Gin Glu Arg Leu Arg Ser Lys

TCC TTG TTC TTA TCA ACA AAQ ATG CAA GAA AGA CTG AGA ACT AAA 478

170 Asp Arg Asp Leu Gly Ser Ser

GAT AGA GAC CTG GGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA 530

11157241912

TAACACTTGCACATGTG ACTCTGGTCAATTCAAAACACTCTTATTTCGGCTTTAATCAC 589

AGAATTGACTGAATTAGTTCTGCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA 648 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT 707 T1ATTCTTACATTTTATCATTTTATACTATTTATATTCTTATATAACAAATGTTTGCC 766

TTTACATTGTATTAAGATAACAAAACATGTTCAGct802

Alul

The insert fragment is further digested with the restriction endonuclease Sau3A and

To obtain a fragment of the desired length of 189 lbf plasma fragment

du P9A2 is digested with restriction and isolated by chromatography

endonuclease Avail. After chromo- and electroelution. It has

matography and purification of the obtained cDNA - the following structure:

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Asp Leu Pro Gin Asn His Gly Leu Leu Ser Arg Asn Thr Leu

V

Sau3A GAT CTG CCT GAG AAC CAT GGC CTA CTT AGC AGG AAC ACC TTG 42

20 NC°T 2530

Val Leu Leu His Gin Met Arg Arg lie Ser Pro Phe Leu Cys Leu

GTG CTT CTG CAC CAA ATG AGG AGA ATC TCC CCT TTC TTG TGT CTC 87

354045

Lys Asp Arg Asp Phe Arg Phe Pro Gin Glu Met Val Lys Gly

AAG GAC AGA AGA GAG TTC AGG TTC CCC CAG GAG ATG GTA AAA GGG 132

505560

Ser Gin Leu Gin Lys Ala His Val Met Ser Val Leu His Glu Met

t ACG CAG TTG CAG AAG GGC CAT GTC ATG TCT GTC CTC CAT GAC ATG 177

Leu Gin Gin He

CTG CAG CASau3Ag ate189

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The fragment with a plasmid (initiation) codon is then digested twice with ZauiZA in front of pER 33. The desired fragment of 108 bp in length is obtained by PvuII. The fragment of 389 bp in length is obtained after electrophoresis on an agar (agarose) gel, electroelution, and purification on an Elutip column. It has the following riboflavin structure:

EcoRI ZaiZA

gaattcacgct GATCGCTAAAACATTGTGCAAAAAGAGGGTTGACTTTGCCTTGCCGA 59

ACCAGTTAACTAGTACACAAGTTCACGGCAACGGTAAGGAGGTTTAAGCTTAAAG ATG 116

RBS Hind III

Cys Asp

TGT gate

with TA123

SauJA

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Ligation of fragments b and c is cut with Hind III. This ligated fragment has the following

and after the destruction of the enzyme, the structure is broken down:

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Hind IIIо, 5 m t

ZaiZDNcol

a AGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA50

ACACCTTGGT GCTTCTGCAC CAAATGAGCA GAATCTCCCC TTTCTTGTGT100

CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG150

CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC200

AGATCACACA TCTTTAagct

Sau3A Hind III

This DNA fragment, necessary for obtaining the pRHWIO plasmid, can also be prepared by using two synthetically obtained oligonucleotides:

The oligonucleotide of formula ACCTTAAACATCTCT-3 is left dephosphorylated at its 51-end. The oligonucleotide of formula 5-GATCACACATCTTA-3 is kinased at the 5-end by Tv-polynucleotide kinase and ATP. When both oligonucleotides hybridize, the following short DNA fragment is obtained:

5f- AGCTTAAAGATGTGT 3

3- ATTTCTACACACTAGp 51

Thanks to this, a

typical Hind III 5-junction and at the other end - typical Sau3A 5-junction.

Fragment b is dephosphorylated using calf intestinal phosphatase. Fragment b and the above fragment are assembled together and linked to each other using T-ligase.

Since the ligase requires at least one 5'-phosphate-containing end, it is possible to link only a piece of synthetic DNA to fragment 6 or two synthetic fragments at their Sau 3A ends. Since the two resulting fragments differ in length, they can be separated by selective precipitation with isopropanol. The purified fragment is then phosphorylated by Tu polynucleotide kinase and ATP.

Example 2.

II., Obtaining expression plasmids.

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a. Preparation of plasmid pRHWIO. The expression plasmid pER 103 was linearized with Hind III and then treated with calf intestinal phosphatase. After isolation and purification, the resulting DNA was dephosphorylated and then ligated with the ligated bis fragments (after digestion with Hind III). E. coli HB 101 was then transformed with the ligation mixture and cultured on LB-arape plus 50 μg/ml ampicillin. The plasmid with the desired structure was named pRHWIO, which, after its replication, served as an intermediate for the preparation of further plasmids.

b. Obtaining plasmid pRHW12.

The Klenow fragment of DNA polymerase 1 and 4 deoxynucleoside triphosphate are added to the BamHI-cut pRHWIO plasmid. The linearized and blunt-ended plasmid obtained after incubation is purified and then cut with Ncol. The larger fragment, which is obtained by agarose gel electrophoresis, electroelution, and Elutip purification by elution type, is ligated with fragment a. The ligation mixture is then transformed into E. coli HB 101 and cultured on LB-arape plus 50 μg/ml ampicillin. The plasmid with the desired structure was named pRHW12, 1 liter of the resulting incubated bacterial culture (optical density 0.6 at 600 nm) contains 1-10 international units of interferon.

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V. Preparation of plasmid pRHWl1„ To the plasmid pRHWIO cut with Bam HI, the Klenow fragment of DNA polymerase 1 and 4 deoxynucleoside phosphate are added. The linearized plasmid obtained after incubation, equipped with linear ends, is purified and then cut with Ncol. The larger fragment, which is obtained by electrophoresis in agarose gel, electroelution and Elutip purification (by elution type), is ligated with fragment a, similarly obtained from plasmid E79E9, in which only the codon encoding the third amino acid - (Gly) GGG- is replaced by the GAG-codon (Glu). Then E. coli HB 101 is transformed with the mixture obtained after ligation and cultured on LB-arape. Possessing The plasmid was named pRHWll with the desired structure. pRHWl1 excretes the desired omega-(Gly)-interferon.

Example 1. Finding IFN sequence-specific clones.

a. Preparation of cDNA library. mRNA from virus-stimulated

Sendai cells are used as a starting material for the preparation of a cDNA bank using methods known from the literature. The resulting 30,000 cells are individually transferred into the wells of microtiter plates. The following medium is used for colony growth and freezing: 1 g triglyceride; 5 g yeast extract; 5 g NaCl; 0.51 g sodium citrate X2H2O; 7.5 g KZKP04 2 HZ0; 1.8 g KH4PO4; 0.09 g MgSO4-7H2O; 0.9 g (,; 44 g glycerol; 0.01 g tetracycline UNS1; water to 1 l.

For microtiter assays, plates containing individual clones are incubated overnight at 37°C and then stored at -70°C.

b. Hybridization probe (sample The recombinant plasmid pBR 33 serves as the starting material for the hybridization sample. This plasmid contains the coding region for mature interferon IFN-2 arg plus 190 bases of the 3-untranslated region. 20 μg of peR 33 are incubated for 1 h at 37 °C with 30 units of Hind III restriction endonuclease in

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200 µl of reaction buffer (10 mM Tris-HCl, pH 7; 10 mM MgCl4; 1 mM dithiothreitol (DTT); 50 mM NaCl). The reaction is stopped by adding 1/25 volume of 0.5 M ethylenediaminetetraacetic acid (EDTA) and heated at 70 C for 10 min. After adding 1/4 volume of 5x buffer (80% glycerol; 40 mM Tris-acetic acid, pH 7.8; 50 mM EDTA, 0.05% sodium dodecyl sulfate (SDS); 0.1% bromophenol blue), the resulting fragments are separated by size in 1% agarose gel (gel buffer and electrode buffer (TBE): 10.8 g/L Tris-hydroxymethylaminomethane (Tris base); 5.5 g/L boric acid; 0.93 g/L EDTA). After incubating the gel in ethidium bromide solution (0.5 μg/ml), the gel section containing the DNA fragment with the IFN gene (approximately 800 bp in length) is excised. DNA is electro-eluted in 1/10x TBE buffer. The DNA solution is extracted once with phenol and four times with ether, and the DNA is precipitated by adding 1/10 volume of 3 M sodium acetate (Na AC), pH 5.8, and 2.5 volumes of ethanol at -20°C. The DNA pellet is washed once with 70% ethanol and dried for 5 min under vacuum. The DNA is dissolved in 50 μl of water (approximately 50 μg/μl). This DNA is synthesized by the gap displacement method: 50 μl of the reaction mixture contains 50 mM Tris-HCl; pH 7.8; 5 mM MgCl3; 10 mM mercaptoethanol; 100 ng of pER 33 insert DNA, 16 ng DNaSel; 25 μM dATP; 25 μM dGTP; 25 μM dTTP; 20 μM Cl V -Pj-d CTP (> 3000 Ci/mmol), and 3 units of DNA polymerase 1 (E. coli). The reaction is carried out at 14°C for 40 min. The reaction is stopped by adding 1 volume of a solution containing 50 mM EDTA; 2% SDS; 10 mM Tris-HCl, pH 7.6, and heating at 70°C for 10 min. DNA is separated by chromatography through Sephadex G 100 in TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA) from unincorporated radioactivity. The radioactively labeled sample has a specific activity of approximately 4 10 cpm/μg.

c. Screening of clones for the content of i insertions of the IFN gene.

Bacterial cultures frozen into microtiter plates are thawed (a). A piece of nitrocellulose filter corresponds to 17

of the appropriate size (pore size 0.45 µm) are placed on LB agar (LB araprl0 g/l tryptone; 5 g/l yeast extract; 5 g/l NaCl; 15 g/l bacto agar; 20 mg/l tetracycline HCl). Using a stamp adapted for microtiter plates, individual clones are transferred to a nitrocellulose filter. Bacteria are grown overnight at 37 C until large colonies (approximately 5 mm in diameter) are formed. To destroy bacteria and denature DNA, nitrocellulose filters are placed in turn on a stack of Whatman filters soaked in the following solutions: 1 - for 8 min in 0.5 M NaOH; 2 - for 2 min in 1 M Tris-HCl, pH 7.4; 3 - for 2 min in 1 M Tris-HCl, pH 7.4, and 4 - for

4 min in 1.5 M NaCl, 0.5 M Tris-HCl, pH 7.4. The filters are air-dried and then kept for 2 h at SO°C. The filters are pre-treated for 4 h at 65 C in a hybridization solution consisting of 6x SSC (1x SSC corresponds to 0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0), 5x Denhardt's solution (1x Denhardt's solution corresponds to 0.02% PVP (polyvinylpyrrolidone); 0.02% FicoII (molecular weight 40,000), 0.02% BSA (bovine serum albumin), and 0.1% SDS (sodium dodecyl sulfate). Approximately 1-10 cpm of the sample prepared according to (b) are taken onto the filter, denatured by boiling, and added to the hybridization mixture. Hybridization is carried out for 16 h at 65 C. The filter is washed at 65 C with four washes for 1 h in 3x SSC/01% SDS buffer. The filters are air-dried, coated with a thin film and exposed to Kodak X-Omats film.

Example 2. Southern transfer to confirm the presence of the IFN gene in recombinant plasmids.

From colonies that give a positive signal during radioautography, 5 ml cultures are grown overnight at 37°C in LB medium (10 g/l triton; 5 g/l yeast extract;

5g/l NaCl; 20 mg/l tetracycline

x НС1). Plasmid DNA is isolated according to Birnboim and Doly. 1.5 ml of cell suspension is centrifuged and suspended at 0°C in 100 μl of lysis solution consisting of 50 mM glucose;

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19 18 10 mM EDTA; 25 mM Tris-HCl, pH 8.0, and 4 mg/ml lysozyme. Incubate for 5 min at room temperature, add 2 volumes of ice-cold solution (0.2 M NaOH and 1% SDS), incubate for another 5 min, then add 150 μl of ice-cold potassium acetate solution, pH 4.8, and incubate for 5 min. The resulting precipitate is separated by centrifuge. The DNA solution is extracted with 1 volume part of a mixture of phenol and SNCS (1:1), the DNA is precipitated by adding 2 volumes of ethanol. After sedimentation in a centrifuge, the precipitate in the tube is washed once with 70% ethanol and dried for 5 min under vacuum. DNA is dissolved in 50 μl of (TE) buffer. 10 μl of the resulting solution are added to 50 μl of the reaction buffer (10 mM Tris-HCl, pH 7.5; 10 mM MgCl3; 50 mM NaCl, 1 mM DTT), digested together with 10 units of Pstl restriction endonuclease for 1 h at 37°C. Then 1/25 of the volume of 0.5 M EDTA; 1/4 of the volume of 5x buffer are added, heated for 10 min at 70 C. DNA is separated electrophoretically in 1% agarose gel (TBE buffer). DNA is transferred from the agarose gel to a nitrocellulose filter using the Sauzer method. The DNA in the gel is denatured for 1 h with 1.5 M NaCl/0.5 M NaOH and then neutralized for 1 h with 1 M Tris × HCl, pH 8 (1.5 M NaCl). The DNA is transferred into 10x SSC (1.5 M NaCl; 0.15 M sodium citrate; pH 7.0) onto a nitrocellulose filter. After complete transfer (approximately 16 h), the filter is briefly rinsed in 6x SSC buffer, air dried, and then heated for 2 h at 80°C. The filter is then treated at 65°C for 4 h with 6x SSC/5x Denhardt's solution/0.1% SDS. Approximately 2406 cm of the hybridization probe are denatured by heating to 100°C and then added to the hybridization solution. The hybridization duration is 16 hours at 65 C.

Example 3. Detection of interferon activity in clone E76E9.

100 ml of clone E76E9 culture are grown in LB medium (see example 2) at 37 C to an optical density of A600 - - 0.8. Bacteria are separated in a centrifuge for 10 min at 7000 rpm, washed once

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solution of 50 mM Tris x HCl, pH 8.0, 30 mM DOaCl, suspended in 1.5 ml of washing solution. Incubated with 1 mg/ml lyocyme at 0°C for half an hour, the bacterial suspension is frozen and thawed five times. The remains of destroyed cells are sedimented by centrifugation for 1 hour at 40,000 rpm. The supernatant is sterilely filtered and tested for interferon activation. A reduction test plaque with V3 cells and the Vesicular Stomatis virus are used as a test. The clone produces up to 9,000 IV (international units) of interferon per 1 liter of the original culture.

Example 4. Obtaining the expression plasmid pRHW12 and the expression plasmid pRHWl1.

a. Obtaining the pRHWIO plasmid.

100 μg of plasmid P9A2 are hydrolyzed with 100 units of restriction endonuclease Avail. After hydrolysis, the enzyme is inactivated by heating at 70°C and the resulting fragments are separated in a 1.4% agarose gel with TBE buffer according to size. The band containing the entire cDNA insert is electroeluted and purified on an Elutip column. Of the 20 μg obtained, 6 μg are then hydrolyzed with restriction endonuclease 3a3A (20 units in 100 μl of solution). The fragments are separated in a 2% agarose gel in TBE buffer. After staining with ethidium bromide (EtBr), a 189 bp piece of DNA is electroeluted and purified as described.

In order to link the interferon gene to the promoter, ribosome binding site and start codon, the corresponding piece of DNA is isolated from the expression plasmid pER 33. For this purpose, 50 μg of pER 33 is hydrolyzed twice with the restriction enzymes EcoRI and PvuII, and the resulting fragments are fractionated according to their size in a 1.4% agarose gel in TBE buffer. The 389 bp piece of DNA, which contains the Trp promoter, ribosome binding site and start (initiation) codon, is electroedullated and purified using an Elutip column. The resulting fragment is then hydrolyzed with ZauZA and the desired 108 bp fragment is obtained by electrophoresis in an agarose gel, electrophoresis

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920 elution and purification on an Elutip column with a yield of approximately 100 ng.

20 ng of fragment o are ligated at 14°C for 18 h with 20 ng of fragment c in a volume of 40 µl using

10 units of T4 ligase in a solution containing 50 mM Tris-HCl, pH 7.5;

10 mM MgCl4; 1 mM DTT and 1 mM ATP. The enzyme is then inactivated by heating to 70 C and the resulting DNA is cut with Hind.Ill in a volume of 50 µl.

10 μg of the expression plasmid pER 103 are linearized with Hind III in a volume of 100 μl. The mixture is incubated for 2 h at 37°C, 1 volume of 2-phosphatase buffer (20 mM Tris-HCl, pH 9.2; 0.2 mM EDTA) is added together with 1 unit of bovine intestinal phosphatase (CIP). After 30 min at 45°C, the next unit of CIP is added and the mixture is incubated again for 30 min. The DNA obtained in this way is purified by double phenol extraction, single CHCl extraction, precipitated by adding 1/10 volume of 0.3 M sodium acetate (pH 5.5) and 2.5 volumes of ethanol.

iOO ng of linearized pER 103 and ligated S and C fragments (after Hind III hydrolysis) are added to 100 µl of a solution containing ligase buffer and ligated at 14°C for 18 h using T DNA ligase.

200 μl of competent E. coli HB 101 are mixed with 20 μl of the doping mixture and incubated for 45 min on ice. Then DNA uptake is completely stopped by thermal heating for 2 min at 42°C. The cell suspension is incubated for 10 min on ice and applied to LB arabinamide containing 50 mg/l ampicillin. Plasmid DNA is isolated from the 24 colonies obtained using the method of Birnboim and Doly. After hydrolysis with various restriction enzymes, the plasmid has the desired structure. It was named pRHWIO.

b. Obtaining plasmid pRHW12.

Approximately 10 μg of pRHWIO plasmid was cut with Bara HI, then the Klenow fragment of DNA polymerase 1 and 4 deoxynucleoside triphosphate was added and incubated for 20 min at room temperature. The resulting linearized blunt-ended plasmid was purified by phenol extraction and alcohol precipitation. It was then cut with the restriction endonuclease NcoI at

21

in a volume of 100 μl. The larger fragment was obtained by agarose gel electrophoresis, electroelution and purification on Elutip. The fragment was obtained by hydrolysis of 4 μg of the Avail fragment, which contains the P9A2 cDNA insert, with Ncol and Alul, yielding approximately 2 μg of the fragment.

In the last step of ligation, fragment a and the doubly digested BamHI/Ncol pRHWIO added to it are ligated in a volume of 10 µl, with 10 ig of each DNA being used. Ligation of the completed BamHI restriction site Alul again yields a BamHI restriction site.

The mixture obtained after ligation is used to transform competent cells of E. coli HB 101 in the same way as described. One of the 40 colonies obtained is selected and named pRHW12.

The plasmid was isolated and the EcoRI/BamHI insert was sequenced by the dideoxy method. The plasmid had the expected sequence.

c. Obtaining the plasmid pRHWl1. Carried out similarly to the example

46. 1 μg of pRHWIO plasmid is hydrolyzed with BamHI. The sharp ends of the resulting DNA are made blunt using the Klenow fragment of DNA polymerase G and 4 deoxynucleoside triphosphates, then the linearized DNA is cut with Ncol. The larger fragment is obtained by electrophoresis in agarose gel, electroelution, and column chromatography by elution type.

The NcoI-AluI fragment is isolated from clone E76E9 similarly to Example 4b. Then 10 ng of the vector part are ligated with 10 ng of the cDNA part in a volume of 10 μl under appropriate conditions and using 1 unit of T4 ligase. The resulting DNA mixture is used to transform E. coli HB 101. A clone designated pRHWII is selected by selection of the resulting 45 colonies on ampicillin-containing LB plates. The corresponding clone is grown and plasmid DNA is isolated. Its structure is confirmed by the presence of several specific restriction endonuclease cut sites (Alul, EcoRI, HindiII, Ncol, PstI).

g. Expression of interferon activity by E. coli KB 101 cells containing the pRHW12 plasmid.

about 5

0

5

0

Q

with Q

5

1922

100 ml of bacterial culture are incubated to an optical density of 0.6 at 600 nM in minimal medium M9, which contains all amino acids except tryptophan (20 μg/ml per amino acid); 1 μg/ml thiamine; 0.2% glucose and 20 μg/ml indole-(3)-actylic acid (IAA), an inducer of the tryptophan operon. Then the bacteria are precipitated by centrifugation (10 min at 7000 rpm), washed once with 50 mM Tris-HCl buffer, pH 8; 30 mM NaCl, and suspended in 1.5 ml of the same buffer. Incubate for 30 min with 1 mg/ml lysozyme on ice, the bacteria are frozen and thawed 5 times. Cell debris is removed by centrifugation for 1 h at 40,000 rpm. The supernatant is sterile filtered and tested for interferon activity in a spot reduction assay using human A549 cells and Encophalomyocarditis virus.

Result: 1 liter of prepared bacterial culture contains 1-10 international units of interferon.

Claims (1)

Formula of invention  A method for producing recombinant plasmid DNA pRHWl1 or pRHW12 encoding omega-interferon, comprising treating plasmid P9A2 or E79E9 with endonuclease Avail, isolating the cDNA insert, treating the resulting fragment with endonuclease Sau3A, followed by isolating a 189 bp fragment, treating the plasmid with enzymes EcoRI and PvuII, treating the resulting 389 bp fragment containing the trp promoter, ribome binding site and start codon with endonuclease Zau3A, isolating a 108 bp fragment, ligating the resulting fragment with the 189 bp fragment. using DNA ligase, treatment of the obtained plasmid DNA with the endonuclease Hindlll, dephosphorylation, ligation of the obtained fragments, transformation of the bacterial strain E. coli KB 101 with recombinant plasmid DNA, culturing the strain, isolation of the plasmid pRHWIO, treatment of the plasmid pRHWIO with the endonuclease Bam HI, incubation in the presence of the Klenow fragment of DNA polymerase 1 and four deoxynucleases23,157241924 103 contains a DNA fragment with the following nucleotide sequence: Hindlll SauA Ncol aAGCTTAAAG ATGTGTGATC TGCCTCAGAA CCATGGCCTA CTTAGCAGGA50 ACACCTTGGT GCTTCTGCAC CAAATSAPPA GAATCTCCCC TTTCTTGTGT100 CTCAAGGACA GAAGAGACTT CAGGTTCCCC CAGGAGATGG TAAAAGGGAG150 CCAGTTGCAG AAGGCCCATG TCATGTCTGT CCTCCATGAG ATGCTGCAGC200 ACATCACACA TCTTTAagctt SauSA Hindlll ligation of the obtained fragment with the NcoI-AlulI fragment of the P9A2 plasmid with the following nucleo- or E79E9 plasmid sequence: CAT GGG CTA CTT AGC AGG AAC ACC TTG 28 Ncol GTG CTT CTG CAC CAA ATC AGG ASA ATC TCC CCT TTC TTG TGT CTC 73 AAG GAG AGA AGA GAG TTC AGG TTC CCC CAG GAG ATG STA AAA GGG 118 AGC CAG TTG CAG AAG GCC CAT GTC ATG TCT GTC CTC CAT GAG ATG 163 CTG CAG CAC ATC TTC AGC CTC TTC CAC ACA GAG CGC TCC TCT GCT 208 GCC TGG AAC ATG ACC CTC CTA GAG CAA CTC CAC ACT GGA CTT CAT252 CAG CAA CTG CAA CAC CTG GAG ACC TGC TTG CTG CAG GTA GTG GGA298 GAA GGA GAA TCT GCT GXG GCA ATT AGC AGC CCT GCA CTG ACC TTG343 AGG AGG TAG TTC CAG GGA ATC CGT GTC TAG CTG AAA GAG AAG AAA388 TAG AGC GAG TGT GCC TGG GAA GTT GTC AGA ATG GAA ATC ATG AAA433 TCC TTG TTC TTA TCA ACA AAC ATG CAA GAA AGA CTG AGA ACT AAA478 GAT AGA GAG CTG CGC TCA TCT TGAAATGATTCTCATTGATTAATTTGCCATA530 TAACACTTGCACATGTGACTCTGGTCAATTCAAAAGACTCT TATTTCGG.CTTTAATCAC589 AGAATTGACTGAATTAGTTGTCCAAATACTTTGTCGGTATATTAAGCCAGTATATGTTA648 AAAAGACTTAGGTTCAGGGGCATCAGTCCCTAAGATGTTATTTATTTTTACTCATTTAT707 TTATTCTTACATTTTATCATTTTATACTATTTATATTCTTATATAACAAATGTTTGCC766 TTTACATTGTATTAACATAACAAAACATCTTCAGct802 Alul it where X is a C or A nucleotide, recombinant plasmid DNA with postchromatograph, ligation and transfer, followed by isolation of the target product, E. coli bacterial strain HB 101 so Compiled by N. Kuzenkova Editor M. Petrova Technical editor M. Didyk Proofreader N. Korol Order 1524Circulation 498Subscription RCIIPI of the State Committee for Inventions and Discoveries under the USSR State Committee for Science and Technology 113035, Moscow, Zh-35, Raushskaya emb., 4/5 Production and publishing plant Patent, Uzhgorod, Gagarin str., 101