SU1479005A3 - Method of producing human interleukin - Google Patents

Abstract

PURPOSE:To obtain the title polypeptide free from other proteins originating from human and from saccharides by culturing procaryotic cells transformed with a specific recombinant DNA. CONSTITUTION:Procaryotic cells, preferably in Escherichia coli are transformed with a recombinant DNA which includes the genes coding (a) a promoter, (b) a ribosome-binding site, (c) a translation-starting codon and (d) a polypeptide having human interleukin 2 activity in this order from the upperstream of plasmid vector DNA chain which is compatible to procaryotic cells, and cultured aerobically in suspension.

Description

one

The invention relates to genetic engineering and relates to a method for producing human interleukin-2 in cells of Escherichia coli bacteria.

The aim of the invention is to increase the yield of interleukin-2.

The increase in protein yield in E. coli cells is ensured by constructing recombinant plasmid DNA pTI oL2-22, pTU-Ioi2-22, pGI, 2-22, pTI oi2-21a, pTI oi 2-21b, encoding the synthesis of interleukin- 2, the plasmids obtained transform E. coli bacterial cells.

Example 1. A human leukemia T cell line, Jurkat cells are suspended in RPMI 1640 medium containing 10% (v / v) FCS, and receive

X-rays up to 10,000 R. at room temperature for 50 s. Then the cells subjected to irradiation are cultured for 5 days at 37 ° C in an incubator containing 5% C0a, with an initial cell density of Ixl0ff cells / ml. The cells after treatment with mutagen (0.2 cells / recess) are placed in 10 microplate recesses with a flat bottom, each microplate having 96 recesses, and cultured at 37 ° C in an incubator containing 5% CO2 for 21 days. Clones obtained in depressions and showing a tendency to grow are re-transferred to a fresh culture medium. In order to increase the size of the clone and the propagated clones cultivate 1

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for 24 hours at an initial cell density of 1x106 cells / ml in the presence of 50 µg / ml KOH A, the activity of interleukin-2 (IL-2) is measured.

A human T cell line, designated as Jurkat III (J-III) and cloned from the parent cell Jurkat, is then isolated. As a result, the yield of IL-2 is increased 40-fold compared with the parent cells. Line I-III of cloned cells is grown under known conditions, and their growth rate is almost the same as that of ordinary Jurkat cells.

Cells (1x10 cells / ml) of J-III are transferred into 1000 ml of synthetic culture medium, RITC 55-9 in a roller flask for growing crops and cultured for 4 days at

37 ° C, the cells after multiplication are harvested by centrifugation. The harvested cells are again transformed into medium to which 25 µg / ml of KOH A is added so that the cell density is 4x10 cells / ml. Four roller flasks were used for growth, with 1000 ml of the grafted culture medium being loaded into each flask. Cultivation continued for 6 hours while rotating the flasks.

Jurkat cells (1.2x10), stimulated with 25 µg / ml KOH A for 6 hours, are suspended in 8000 ml of phosphate buffer, balanced with brine. The cells are washed twice by centrifuging: and resuspended in 800 ml of RSB solution (10 mmol Tris-HCl, pH 7.5; Humol / NaCl; 1.5 mmol MgCla) containing the Rinucleoside-Vanadil complex (UmM) as a nuclease inhibitor . Then, NP-40 detergent is added so that its final concentration is 0.05%; the contents are vigorously stirred, and the cell nuclei are removed by centrifuging for 5 minutes at a speed of 3000 rpm at. SDS (0.5%) and EDTA (5 mmol) are added to the upper layer and cytoplasmic RNA is extracted by adding an equal volume of phenol. After extraction with phenol three times, the RNA is precipitated with two volumes of ethanol and the precipitates are collected by centrifugation, then dissolved in 10 mmol Tris-HCl at pH 7.5. The amount of RNA obtained is 196 mg.

Fractionation of mRNA was performed using chromatography on oligo (dT) -cellulose. The absorption solution is a solution at pH 7.5, containing 20 mmol of Tris-HCl, 0.5 mol of NaCl, 1 mmol of EDTA and 0.5% SDS, and the elution is carried out with water and 10 mmol of Tris-HCl (pH 7, 5) in turn after washing the column with buffer (20 mmol Tris-HCl, pH 7.5; 0.5 mmol NaCl; 1 mmol EDTA). As a result of elution, 3.6 g of mRNA is obtained. Then, 2.4 mg of the obtained mRNA is fractionated by centrifuging in a gradient of sucrose density (sucrose gradient from 5 to 2.5% in a solution with pH 7.5, containing 50 mmol Tris-HCl, 1 mmol EDTA and 0, 2 mol aCl ), subjected to centrifugation at a speed of 26,000 rpm for 24 hours at 4 ° C, and fractions from 11 to 12C RNA is subjected to fractionation into ractions No. 12, 13 and 14 in an amount of 59; 46 and 60 μg, respectively.

The RNA obtained in fraction No. 13 is watered in the form of a microinjection in the ovocyte Xenopus lalvis (50 ng mRNA / cell) and the culture top layer is used to analyze the activity of IL-2.

As shown in the table. 1, an increase in the received dose of 3H-TDD and an increase in the number of activated T-lymphocytes is consistent, which indicates that the mRNA in this fraction contains human IL-2 mRNA.

Next, cDNA is synthesized in laboratory conditions from fraction No. 13 with P-12C mRNA containing IL-2 mRNA, and recombinant DNA is constructed using the plasmid pBR 322 vector. Escherichia coli and a clone containing IL clones are encouraged with recombinant DNA. -2 cDNA, is isolated as follows: 50 mmol of Tris-HCl buffer (pH 7.5), 30 mmol of NaCl, 6 mmol of MgCl., 5 mmol of dithiothreitol (DTT), 0.5 mmol of each of dATP, dGTP, dPTP, dTTP (dCTP g consists of radiolabeled 32P dCTP), 0.7 µg oligo (dT) 10, U µg mRNA and I 5 units, AMV reverse transcriptidase is mixed and maintained in those ix 90 min at 41 C.

After the completion of the reaction, the DNA is extracted as ethanol precipitates after treatment with phenol, and then the DNA is dissolved at pH 7.5 in solution,

containing 20 mmol Tris-HCl and 1 mmol EDTA. As a result, 2.5 μg of he-cDNA is synthesized. To remove the mRNA contained in this solution, prepare 0.33 n. NaOH solution by adding NaOH, then the solution is kept for 15 hours at room temperature and neutralized with an equal volume of 1 M Tris-HCl at pH 7.5. Next, the resulting solution is passed through a column of Sephadex G-50. A total of 1.8 μg of cDNA is obtained.

50 mmol phosphate buffer, pH 7.5, 10 mmol MgCl2, 10 mmol DTT, 0.75 mmol each of dATP, dGTP, dCTP, dTTP (dCTP consists of radiolabeled EN dCTP), 1.8 μg of he-cDNA and 8 units. Polymerase I is mixed for 15 hours at 15 ° C. After completion of the reaction, the DNA is recovered as an ethanol precipitate after several treatments with phenol and chloroform. Forms 1,10 µg dn-cDNA. A mixture of 50 mmol of sodium acetate (pH 4.5), 0.2 mmol of NaCl, 1 mmol of ZnCla and 1.10 g of day-cDNA are incubated for 20 minutes at 37 ° C, then 0.25 units are added to it. S nucleases and the mixture is incubated for another 15 minutes.

After completion of the reaction, the resulting product; twice treated with phenol, passed through Sephadex G-50, resulting in a gain of 0.55 μg day-cDNA.

A mixture of 0.14 mol of potassium cacodylate,

30 mmol of tris-base, 0.1 mmol of DTT, 1 mmol of CoCl2, 0.64 mmol of 32 P-dCTP (specific activity 2.7x10 counts min nmol), 0.55 μg of day-cDNA and 5 units. the terminal transferase is incubated for 7 minutes at 37 ° C, then passed through Sephadex G-50 after treatment with phenol to obtain 0.50 μg of DNA in the form of ethanol precipitates. The extracted DNA contains approximately 50 dCMP residues at both 3 terminals.

10 μg of the plasmid pBR 322 DNA is digested with an enzyme of the Pst I section and to

The 31 terminals of the dissected DNA are added with a dGMP chain using the same method as when adding dCMP to the day cDNA, except that dGTP is used instead of dCTP.

A mixture of 50 mmol Tris-HCl (pH 7.5), 0.1 mol NaCl, 5 mmol EDTA, 0.05 micron pBR 322, extended by dGMP residues, and 0.01 g cDNA extended by dCMP, are first incubated for 2 minutes at 65 ° C, then 120 minutes at 46 ° C, 60 minutes at 37 ° C and 60 minutes, at room temperature.

E. coli X 1776 inoculate in 50 ml of L-broth containing 100 μg / ml of diaminopimelic acid, 50 μg / ml

0 thymidine, 1% tryptophan, 0.5% yeast extract, 0.5% NaCl and 0.1% glucose, and cultured with shaking at 37 ° C until the absorption of the culture fluid in the region

5,562 nm will not give an optical density of 0.3. After the cultivation is completed, the culture fluid is maintained at 0 ° C for 30 minutes, then the bacterial cells are harvested using

0, centrifuged, washed twice with 25 ml of a solution containing 5 mmol of Tris-HCl (pH 7.6), 0.1 mol of NaCl, 5 mmol of MgCl2 and 10 mmol of RbCl.

Thus obtained cells

5 are suspended in 20 ml of a solution containing 5 mmol of Tris-HCl (pH 7.6), 0.25 mol of KCl, 5 mmol of MgCla, 0.1 mol of CaCl2 and 10 mmol of RbCl, and kept at 0 ° C for 25 minutes.

Next, cells are harvested and resuspended in 1 ml of the same solution. Then the recombinant DNA described above is added to 0.2 ml of the cell suspension and the suspension is kept at 0 ° C for 60 minutes. The culture is added

O, 7 ml of L-broth and the mixture is incubated with shaking for 30 minutes at 37 C. The culture medium thus obtained (O, 1 ml) is equal to

Q is evenly distributed on the surface of a medium containing 1.5% agar consisting of L-broth containing 100 µg / ml of diaminopimelic acid, 50 µg / ml of thymidine and 15 µg / ml

5 tetracycline, and incubated at 37 ° C for 2 days.

The resulting 432 colonies are divided into 18 groups, each of which contains 24 different bacterial thermal clones, inoculated into 200 ml of L-broth containing 100 µg / ml of aminopimelic acid, 50 µg / ml of thymidine and 10 µg / ml of tetracycline, and cultured with shaking when

5 to 37 ° C for 5 to 7 hours. Then, 200 ml of fresh L-broth containing 170 µg / ml of chloro-amphenicol with a concentration of 170 µg / ml is added to the culture and the cultivation is continued overnight.

The DNA plasmid thus obtained after amplification is purified using known means. Clones containing IL-2 cDNA

are selected using the analytical method of hybridization - translation of mRNA (G - T-analysis).

The RT analysis is carried out as follows.

Purified DNA (25 µg) is cut with the Hind III restriction enzyme, treated three times with phenol, phenol-chloroform and chloroform, respectively, precipitated with ethanol, washed with ethanol (80%) and dissolved in 40 µl of 80% formamide.

Then the reaction mixture is heated

in order to denature to a temperature of 90 ° C, which is maintained for 5 minutes, the potency of IL-2. Therefore, this clone

As shown in the table. 2, only the DNA plasmid obtained after purification from a single colony, designated p3-16, gives a positive ak

diluted with 1.3 ml with 10 x SCC (1.5 mol} NaCl, 0.15 mol sodium citrate). Next, the DNA is fixed on nitrocellulose filters, the filtrates are dried at up to 80 ° C for 3 hours and incubated for 18 hours at 37 ° C in a solution containing 50% formamide, 20 mmol Pipes (pH 6.5), 0.75 mol NaCl, 5 mmol EDTA, 0.2% SDS, and 250 µg of poly (A) mRNA obtained from J-III induced cells to obtain a DNA hybrid fixed on the filters with IL-2 mRNA. Then the filters are washed with 65 C three times with a solution containing 10 mmol Pipes (pH 6.5), 0.15 mol NaCl, 1 mmol Pipes, 10 mmol NaCl, and treated with 0.5 mmol EDTA, 0.1% SDS solution at 95 ° C for 1 min in order to extract with f ltrov gibridizi- Rowan. mRNA. The mRNA extracted in this way is purified on an oligo-dT-cellulose column in accordance with known methods and injected into Xenopus oocytes to determine the activity of IL-2 translated proteins. One of 18 groups, each of which consists of 24 clones, gives a positive IL-2 activity of 48 units / ml in the H-TdR absorption assay; while other groups give a clear negative result.

Then 24 individual colonies are identified as a clone containing IL-2 cDNA (E. coli X1776 / r3-16, A T 11995 (FERM-BP-225). Thus, the DNA plasmid p3-1 6 does indeed contain DNA (gene IL-2) capable of forming a special hybrid with IPiRNA.

The cDNA insert of the p3-16 plasmid has properties such that psz

30 will dissect it with the Xbal section enzyme in a single site, and the BstNI enzyme in two sites (up and down from the Xbal section site). However, plasmid rz-16 contains cDNA insert

35 consisting of approximately 650 base pairs, which corresponds to a part of IL-2 mRNA from 11-12C.

Therefore, another cDNA library is prepared using IL-2 mRNA as

40 template. Single strand cDNA

(1.6 μg) is synthesized using 4 μg of IL-2 mRNA, extended with dCMP residues of a day-cDNA, and synthesized using oligo

45 (dG) 12 ..- 18 as a primer for DNA polymerase I (Klenow fragment of the CNDNA (0.6 µg), exceeding the DNA size marker in 680 pairs of innovations, is obtained by centrifuging with a sucrose gradient and inserting the plasmid pBR322 is sent to the Pst I site using the standard GC-cut method. After transformation of E. coli X 1776 with p

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Approximately 2000 colonies are selected for the positive group when combined with DNA using the in situ hybridization method with the translated ends of the p3-16 cDNA insert as a probe, a colony is detected, mixed in 200 ml of L-broth, cultured under aerobic conditions x for 5-7 hours at 37 ° C and fresh L-broth containing chloramphenicol is added.

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After amplifying the DNA plasmid during the overnight culture, the plasmid is purified according to standard procedures. After dissecting approximately 5 μg of each DNA plasmid with Hind III, each DNA plasmid is fixed on nitrocellulose filters. 0 Filters are hybridized with IL-2 mRNA and hybridized mRNA is extracted, and then injected as an injection into the Xenopus ovocyte in order to determine the activity of IL-2 translated proteins.

As shown in the table. 2, only the DNA plasmid obtained after purification from a single colony, designated r3-16, gives a positive identification as a clone containing IL-2 cDNA (E. coli X1776 / rZ-16, A T 11995 (FERM-BP-225) Thus, the DNA plasmid p3-1 6 actually contains DNA (the IL-2 gene) capable of forming a special hybrid with IP2 mRNA.

The cDNA insert of the plasmid r3-16 possesses such properties as psz

0 they will dissect it with the Xbal cross section enzyme in a single site, and the BstNI enzyme from two sites (up and down from the Xbal cross section site). However, plasmid rz-16 contains a cDNA insert,

5 consisting of approximately 650 base pairs, which corresponds to a part of IL-2 mRNA from 11-12C.

Therefore, another cDNA library is prepared using IL-2 mRNA as a template. Single strand cDNA

(1.6 µg) is synthesized using 4 µg of IL-2 mRNA, extended with dCMP residues, and the d-cDNA is synthesized using oligo

5 (dG) 12 ..- 18 as a primer for DNA polymerase I (Klenow fragment), cNDNA (0.6 µg), longer in length than the DNA size marker of 680 base pairs, is obtained by centrifuging a sucrose gradient and inserting to the Pst I site of the plasmid pBR322 using the standard GC terminus cut-off method. After transformation, E. coli X 1776 using pe0

Approximately 2000 colonies were harvested from the combinant DNA using the in situ hybridization method with the translated ends of the p3-16 cDNA insert as a probe, and a colony was detected.

holding the plasmid p ob2-LOA, 850 base pairs in size (E. coli X17 / 6 / pIci2-50A, Aob 11996 (FERM-BP-226) .5

In order to isolate the gene encoding the Sh1-2 peptide, the plasmid DNA was treated with the Pst I restriction enzyme from the transformed E. coli X 1776 pi-2-50A culture, and the DNA fragment fragment was isolated from cells using known methods. The smaller fragment thus obtained among the two DNA fragments is the DNA gene encoding the IL-2 peptide.

Example 2. The pKCR plasmid consists of the SV 40 virus DNA segments containing the promoter of the original gene, the beginning of replication (0.725 - 0.648 me). polyadenylation site 20 from the original gene (O, 169-0,144 mE part of the rabbit β-globin gene (fragment BamHI-PVU II), a segment from pBR322 (fragment EcoRI-BamHI) containing the start of replication and the resistance gene k25 ampicillin. This plasmid dissected with BamHI and after filling both ends of the cut DNA with DNA polymerase I (Klenow fragment), synthetic DNA of the Pst I linker was inserted in order to complete the construction of pKCR (Pst I). -Plasmid pKCR (Pst I) was extracted with the enzyme Sail , treated with a fragment of maple in order to fill the ends, and then partially dissected with the enzyme EcoRI in order To obtain the EcoRI-Sall fragment, which contains all the DNA obtained from the SV 40 virus, and the globin gene. This fragment is then ligated with a piece of pBR328 DNA, which contains the tetracycline resistance gene and the start of replication. The resulting plasmid pCE-1 contains the only downstream Pst I site 45 immediately after the SV 40 promoter obtained earlier.

The cDNA plasmid pIoi2-50A insert is cut by dissecting with the Pst I enzyme and ligated with pCE-1, 50

dissected by Pst I, in order to construct pCEIoi-2, in which the expression of the structural gene IL-2 should be carried out under the control of the previously inserted SV 40 promoter. Plamid pCE-155 was previously built for cloning. cDNA by the G-C-terminus in bacteria and direct expression in mammalian cells.

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portions of the plasmid for the expression of COS-7 cells (6 x 104 / ml) the suspension This plasmid is treated with Hhal and then introduced using DNA transfection into the COS-7 line of transformed monkey cells, which allows for the replication of DNA containing the initial SV 40 virus sequences.

An important step is the processing of the plasmid with Hhal before transfection to increase the expression efficiency of the cDNA, since sequences that would prevent DNA replication after transfection into COS cells are removed from the essential cDNA.

Penned out in 0.5 ml of DMEM containing 5% F1S, which are placed on a culture plate with 24 wells (Nunk type), and incubated for 4 hours at 37 ° C. Then a mixture of 1 μg of the vector described above, 17.6 μl of 1 mM Tris-HCl containing 0.1 mmol of EDTA, 2.4 μl of 2M CaClg and 120 μl of 2 x HBS containing 50 mmol of Pipes, 280 mmol of NaCl and 1, 5 mmol x 12 (pH 7.10) are added to cultured cells, which are incubated for 4 hours at 37 ° C. The culture medium is dehydrated. The cells are then washed with 1 ml of PBS and cultured in 1 ml of DMEM containing 5% FCS. Every 24 hours, 500 ml of medium are replaced with fresh medium. Each portion of the medium collected by B at a specific point in time is stored at 4 ° C until use. 2-3 days after transfection, the culture medium is analyzed for human IL-2 activity.

As shown in the table. 1, the resulting upper layer of a culture of COS-7 cells after transfection using pCE1e6 2, possesses IL-2 activity. However, no activity of IL-2 was detected in the cell culture medium after transfection with the use of pCE-1.

The IL-2 activity, detected in the cell culture medium after transfection of COS-7 cells with pCEIoi-2, neutralizes monoclonal anti-IL-2 human mouse antibodies (BALB / k). The fact that COS-7 cells after transfection using pCEIoi-2 secrete human IP-2 shows that eukaryotic cells transformed with recombinant DNA containing the gene,

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the encoding polypeptide IL-2, and a DNA vector capable of replication in these cells, can be used to produce IL-2,

Example 30 Escherichia coli X 1776 / pI (x, 2-50A, AT 11,996 / FERM-BP-226 are inoculated into 250 ml of L-broth containing 100 μg / ml of diamicopimelic acid, 50 μg / ml of thymidine, 1% tryptophan , 0.5% yeast extracts, 0.5% NaCl and 0.1% glucose, and cultured with shaking at 37 ° C until an optical density in the region of 562 nm is reached with culture medium 0.5. After the cultivation is completed, the culture medium is maintained at 0 ° C for 30 minutes and the cells are collected by centrifuging, washed once with 20 mmol of Tris-HCl at a content of 30 mmol of NaCl and resuspended in 1.8 ml Buffer. A solution containing 0.2 µl of lysozyme (10 mg / ml) and 20 ml of 0.5 M EDTA is added to the - cells and the mixture is kept at 0 ° C for 20 minutes and then frozen - thawed 3 sequentially. Next, the cell extracts (1.5 ml) are obtained after centrifuging at 40,000 rpm for 30 minutes.The extract is salted out with an 85% ammonium sulfate solution, passed through Sephadex G-15 to remove salts , then placed on a column of chromatography on DEAE-cellulose and the fraction eluted with 0.06 M Tris-HCl buffer ( H 7.6) was collected. The fraction thus collected was freeze dried and placed on glass beads with a specific pore size of 250 A contained in a chromatography column to analyze the activity of IL-2 in the eluent using 0.3 M glycine-HC1 buffer. The fraction containing IL-2 has an activity of IL-2 12 units / ml. The results indicate that E.Coli X1776 / pIoi2-50A, AT 11996 does produce IL-2.

Example 4. A constitutive IL-2 producer T-A1886 cell line (ATCC CRL 8130) is grown in the same manner in a culture culture flask. Grown cells are suspended in fresh synthetic medium RITC-55-9, with an initial cell density of 1x10 cells / ml and release 7900512

8 hours after the start of culture, the cells are used to extract IL-2 mRNA in the form of a fraction of 11-12C.

Synthesize a double-stranded cDNA, and

cDNAs longer than 600 base pairs (2.4 µg) are obtained after fractionation using a sucrose density gradient, cDNA

10 is then extended using dCMP residues using terminal deoxynucleotidyl transferase (50 ng), it is renatured with 250 mg of plasmid pBR322 extended by

15 dGMP and dissected with Pst I. The resulting hybrid plasmids are used to transform E. coli X 1776, resulting in approximately 4,000 transformed clones. Three clones complementary to the cDNA plasmid p3-16 are selected, which is used as a probe.

Example 5. Plasmid which

25 encodes the synthesis of human IL-2 in E. coli Escherichia coli, is constructed as follows. Plasmid 2-22 is constructed from pTr S-3 and pIoi2-50A containing IL-2 cDNA. Plasmid pTr S-3

30 contains the insertion of the Trp and Shine Dalgarno promoter region (Shine Dalgarno - SD) between the EcoRI site and the Clal site of the plasmid pBR 322. In addition, this plasmid contains an ATC codon of 13 bp.

35 located downstream of the SD sequence, as well as the only Sph I site. This vector is effective for producing the indicated J protein if the DNA sequence 40 corresponding to the indicated protein is inserted into the region downstream immediately after ATC codon, which is formed as a result of SphI fermentation and subsequent

45 processing using DNA polymerase T4 plasmid pTr S-3. The pTr S-3 plasmid (30 µg) is cut with Sph I cross section enzyme by known methods and after a series of treatments with phenol and chloroform

50 ethanol precipitates are recovered, then both ends are injected by treatment with T4 DNA polymerase. Next, DNA (21.4 μg) is extracted by analogous treatment with phenol, chloroform

55 MOM and ethanol precipitation. On the other hand, 380 μg of the plasmid ploi 2-50A, containing the cDNA of IL-2, are cut with the help of the enzyme Pst I and IL-2, the cDNA insert is isolated by agar gel electrophoresis. The cDNA insert (11 μg) was cut with Hgi AI, treated with TA DNA polymerase, and 10 μg of large-sized DNA was isolated by agar gel electrophoresis. A cDNA (7.2 μg) containing information on the synthesis of 132 amino acids is obtained, and this DNA fragment has blunt ends.

Then, the thus obtained cDNA fragment is stitched with the pTr S-3 vector, previously fermented using Sph I and treated with T4 DNA polymerase, at a point in the downward direction immediately after the ATC sequence. The plasmid thus coupled is then used to transform E. coli HBIOI cells by known methods.

The crosslinking is carried out as follows. A longer fragment of IL-2 cDNA (0.4 μg) and 0.2 μg of the pTr S-3 vector DNA is mixed with 0.8 units. T4 DNA ligases in 66 mmol Tris-HCl at pH 7.5 with a content of 6.6 mmol MgCl ,, 1 mmol ATP and 10 mmol DTT. The mixture was allowed to react at 4 ° C overnight. Among the transformed culture formed on an agar plate with ampicillin-containing L-broth, colonies are selected that contain a batch of IL-2 cDNA, which includes information on 132 amino acids. Such a selection of osu-. are in situ by an analytical method of colony hybridization. The colonies thus selected are cultivated (10 ml) to obtain plasmid DNA as a result of lysozyme treatment and freeze-thawing. DNA plasmids are cut with Pst I and Xbal and the resulting products are analyzed by agar gel electrophoresis to identify pT1ob2-22, in which the cDNA is cross-linked with the ATC sequence of the pTr S-3 plasmid with the correct orientation . E. coli HBIOI containing pTIoi-2-22 is grown under standard conditions that are used to propagate microorganisms. Cells are grown in 10 ml of X-broth (2.5% bactotrypton, 1.0% yeast extracts, 0.1% glucose, 20 mmol MgS04, 50 mmol tris-HCl, pH 7.5) containing 25 μg / ml streptomycin

and 2j μg of ampicillin at 37 ° C overnight.

A suspension with a volume of 1 ml of culture is grafted in the same broth (100 ml) and the cultivation is carried out at 37 ° C. When the optical density reaches about 1.5-2.0, 3-lidol-acrylic acid (IAA) is added, 3 hours after the addition of the inducer, the cells are harvested, washed with 20 mmol Tris-HCl (pH 7.5; 30 mmol NaCl) and again suspended in 8 ml of the same buffer. For the efficient functioning of the Trp promoter, an inducer such as IAA is added so that its final concentration is 50 µg / ml. The proteins thus obtained in bacterial cells are extracted by sonication (06 ° C, 2 minutes) or by lysozyme fermentation at 8 µg / l (0 ° C, 20 minutes) and freeze-thawing is carried out three times in succession. In accordance with these procedures, IL-2 is generally extracted from organisms. The activity of the extracted IL-2 varies in the range of 10,000 - 120000 units / ml.

Example 6. The plasmid pTUIei 2-22 carrying the IL-2 cDNA is constructed from pTUBIP-5 and pT1 ob2-22. The pT plasmid pTUBIP-5 includes the insertion of the tufB promoter sequence into pBR 322. In addition, the plasmid contains a single Clal site, and it is located down 2 bp. from SD sequence. Since pTr S-3 also contains a Clal site between the SD sequence and an ATC initiation codon, and since this Clal site is not destroyed during the construction of the expression plasmid using pTr S-3 and IL-2 cDNA, the bacterial promoter trp simply replace the tufB promoter so that the IL-2 cDNA is expressed under control of the tufB promoter.

Therefore, the plasmid (30 μg) is cut with restriction enzymes Clal and PVU II using standard procedures. The 2.2 kb fragment containing IL-2 cDNA is isolated and purified by agar gel electrophoresis, resulting in 3 μg of DNA. On the other hand, 20 μg of the pTUBIP-5 vector are similarly dissected with Clal and PVU III and a larger fragment (3.4 kb).

containing the ampicillin resistance gene, is isolated and purified by agar gel electrophoresis to extract 3.5 μg of DNA.

Then, the thus obtained two fragments, one of which (3.4 kb) contains the tufB promoter, and the other (2.2 kb), the IL-2 cDNA, are ligated, which is carried out as follows. The fragment containing IL-2 cDNA (1.2 μg) and 0.3 μg of the fragment containing the tufB promoter are mixed with 0.8 units. DNA ligase T4 in 66 mmol Tris-HCl (pH 7.5) with a content of 6.6 mmol MgCl4, 1 mmol ATP and 10 mmol DTT, and the mixture is allowed to interact at 4 ° C overnight. This plasmid after ligation is used to transform Escherichia coli. E. with 11 HBIOI according to standard procedures. Among the transformed cells formed on an agar plate with ampicillin-containing L-broth, eight colonies were selected containing a portion of IL-2 cDNA, such as pTUIo62-22, and plasmid DNA was obtained. E. coli HBIOI cells containing pTUIo62-22 are cultured in L-broth (100 ml) at 37 ° C. When the optical density in the region of 650 nm reaches about 0.5-1.0, the bacterial cells are harvested, washed with 20 mmol of Tris-HCl (pH 7.5; 30 mmol of NaCl) and resuspended in 2 ml of the same buffer. The proteins thus obtained are extracted. The activity of the extracted IL-2 varies in the range of 6000 - 56000 units / ml.

Example 7. Plasmid pGI 2-22 carrying the IL-2 cDNA was constructed from pGoftOI and.

The pGoilOI plasmid (20 µg) containing the lac promoter is cut with the PVU II cross section enzyme using a standard method to obtain 17 µg of DNA by sequential treatment with phenol, chloroform, and precipitation with ethanol. On the other hand, pT1OЈ2-22 (75 μg) is dissected with Clal and Sail in order to extract 2.2 μg of the DNA fragment containing the cDNA of IL-2 using agar gel electrophoresis. This fragment is supplied with an influx by treatment with DNA polymerase I (Klenow fragment), then the two fragments thus obtained (0.25 μg and 0.66 μg) are ligated with 1.0 units. T4 DNA ligase. The plasmid thus obtained after ligation is then used to transform E. coli HBIOI cells by a standard procedure.

Among the transformed cells are selected cells containing the insertion of a fragment of Clal - Sail, including

cDNA IL-2. These transformed cells are then cultured in X-brothon (10 ml) containing 26 μg / ml ampicillin, and DNA plasmids are obtained. A DNA plasmid containing the ATC sequence initiating downstream of the IL-2 cDNA immediately after the lac promoter is obtained by dissection using Pst I and Xbal enzymes.

0 Thus obtained plasmid pGI bЈ 2-22 is placed in 1 00 ml of L-broth containing 25 μg / ml of ampicillin and 25 μg / ml of streptomycin, and then cultured. When optical density in the region of 650 nm reaches about 0.5, isopropyl-p-D-β-thiogalactopyranoside (IPTG) concentration of 1 mM is added, and after 1 h, the bacterial cells are harvested. The activity of extrapolated IL-2 varies in the range of 6000 - 80000 units / ml.

Example 8. A plamid pTr S-3 (10 µg) is first dissected with the Sail cleavage enzyme and the Sail site is floated by treatment with DNA polymerase (Klenow fragment) or DNA polymerase T4. After dissection with Clal, a larger fragment containing the trp promoter region is

0 is digested by agar gel electrophoresis using standard methods, resulting in 3 μg of DNA.

11 μg of cDNA insert obtained

5, as a result of the Pst I enzyme cross-section, plasmids of p ob2-50 A, dissected with Hgi AI, treated with T4 DNA polymerase, and a larger fragment is isolated and purified using

0 electrophoresis on agar gel. Thus, a cDNA fragment containing information on 132 amino acids of IL-2 is obtained in an amount of 7.2 μg. Then, 0.45 µg of the fragment containing the trp promoter, 0.5 µg of the HgiAI-Pst I fragment containing IL-2 cDNA and synthetic oligonucleotides (5) CG ATAA G CTATGG CA (H) and (3) TATTS G ATACCGT (5 ) each of 20 to 1 mole,

each of which is formed at the 5-terminal, ligated with 1 unit. TA DNA ligase.

The plasmid thus obtained after ligation is then used to transform E. coli HBIOI cells. Among the resulting transformed cells, the desired transformed cells are selected in the following 10 ways.

First, as candidates, transformed cells capable of hybridization with both IL-2 cDNA and synthetic oligonucleotides (this procedure is carried out using the colony hybridization method) are selected. Then, using the Pst I, Xbal enzymes, select the transformed cells containing 20 inserts of the DNA fragment starting with the CST sequence in positions III to 113 of the CCSTT downstream immediately after the AT GG CA follower nose 25

After selection, the transformed cells are cultured with P-broth, while the high activity of IL-2 is established in cell extracts.

The research results are given in tab. 2 and 3.

The host cells E. coli 1776 and BIOI, which have been used, are semi-ene from deposited transformed cells by cultivating 35 transformed cells in

L-broth at 37 ° C to obtain, with the appropriate recombinant DNA in transformed cells, and the separation of strains that become sensitive to tetracycline and ampicillin, as host cells.

Claims (1)

Invention Formula A method for producing human interlenkin-2, which involves culturing cells in a medium followed by isolation and purification of the obtained product g, characterized in that, in order to increase the yield of interlanin-2, Escherichia coli bacterial cell strain is transformed with recombinant plasmid DNA pT1os2-22 or pTUI oU- 22, or pGIot 2-22, or pTIoi, 2-21a, or pTIoi.2-21b, containing an insert encoding interleukin-2. Table I table 2 nineteen Compiled by N. Kuzenkova Editor p. Petrash Tehred L. Serdyukova Korrektor S. Shekmar Order 2378/58 Circulation 501 VNIIPI State Committee for Inventions and Discoveries at the State Committee on Science and Technology of the USSR 113035, Moscow, Zh-35, Raushsk nab. 4/5 Production and publishing plant Patent, Uzhgorod, st. Gagarin, 101 1479005 20 table Subscription