SU1761805A1 - Recombinant plasmid dna pil-2/21, encoding human interleukine-2, method of its construction and strain of bacteria esherichia coli - producer of human interleukine-2 - Google Patents

Abstract

Uses: molecular biologists and genetic engineering. Summary of the Invention: The obtained recombinant plasmid DNA plL-2/21 has a mol. the weight is 2.5 Md and contains the Pstl-Xhol DNA fragment of the plasmid pDST2 + o with the transcription terminator ID and 3 - the terminal part of / 3-lactamase; Pstl-EcoRI the DNA fragment of the plasmid pBR322 with the 5-terminal part of the gene / 3-pac- Tamaz; The EcoRI-EcoRI fragment of the phage M13 grlp with the promoter-operator region of the rec gene A. Proteus mirabilis, as well as the synthetic Haell-SalGI DNA fragment encoding the human interleukin-2 gene, adapted for efficient expression in E.coli cells. Plasmid plL-2/21 was constructed by sequential cloning of fragments in the pBR322 and pDSt + o vectors; the strain producing interleukin-2 was obtained by transforming E. coli SG 20050 cells with this recombinant plasmid. The level of synthesis of interleukin-2 protein in the designed strain is 30-35% of the total amount of cellular protein, which corresponds to 45-50 mg / l (3-4 x 108 IU / l), 3 cf.f-ly. (/

Description

The invention relates to medicine, biotechnology and genetic engineering, and can be used to produce human interleukin-2 protein in E. coli cells.

The aim of the invention is the creation of a bacterial strain-producer of human IL-2, providing a high content of the target protein in biomass.

For this purpose, a recombinant plasmid DNA plL-2/21 has been designed, which allows the expression of the human interleukin-2 gene, consisting of:

-EcoR l-EcoR l-fragment of the DNAphage Mia rpm with the regulatory region of the rec A Proteus mirabilis gene;

DNA fragment with a synthetic human IL-2 gene and an initiating ATG codon;

- plasmid vector, which provides termination of gene transcription and selective selection of clones for ampicillin resistance. To obtain piL-2/21, the synthetic gene of human IL-2 is connected in series with the oligonucleotide. containing the ATG codon, and the regulatory sequence of the hea A sequence from Proteus mirabilis and integrated into the vector plasmid pDSt + o.

The recombinant plasmid DNA pIL-2/21 is transformed into E. coli cells SG 20050, in which the expression of the human IL-2 gene is carried out. The Proteus mirabilis regulatory system is induced by a mitomycin C mitogen. Synthesis of IL-2 protein in cells is fixed by electrophoresis in PAAG, and its identification is carried out with

Xi

with O (L

using enzyme immunoassay and biological activity.

The strain E.coli SG (plL-2/21) is characterized by the following features.

Morphological signs. Straight, rod-shaped cells 1.5x2.0x6.0 µm, motile, with peritrichous flagella, gram-negative, risperadone.

Cultural features. Cells grow well on simple nutrient media. When growing on agar media, colonies form smooth, round, glossy, gray, uneven edge, turbid. With the growth in liquid media, m of septone broth, L-bulsne form a smooth intense slime.

Physiological and biochemical signs. Cells grow in the range of 10-40 ° C at an optimum pH of 6.8-7.6. Many carbohydrates, alcohols, organic acids, in particular, glucose, fructose, lactose, galactose, are used as a carbon source. As a nitrogen source, mineral salts are used in ammonium and nitrate forms and in organic form - peptone, amino acids, nitrates, reduced to nitrates.

Gelatin is not diluted. Urease activity was not detected. Indole do not form.

Antibiotic resistance. They show ampicillin resistance due to the presence of the plasmid plL-2/21, and grow in ampicillin concentrations from 20 to 100 µg-ml,

The strain is deposited in the All-Union Collection of Industrial Microorganisms of the All-Union Scientific Research Institute of Genetics under the number VKM85542.

The level of IL-2 synthesis in a constructed strain is at a culture density of 10 cells / ml of about 50 mg / l or 30-35% of the total amount of cellular protein, which makes it possible to consider the constructed strain as a producer, potentially suitable for the production of IL-2 protein .

PRI me R 1. Construction of recombinant plasmid pBIL-2.

10 μg of recombinant plasmid plL-2 are sequentially hydrolyzed with Hay i I and Sale l restriction enzymes, first in 100 μl of buffer 1 (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM 2-mercaptoethanol, 50 mM NaCI ), and then in buffer 2 (50 mM Tris-HCl pH 7.5, 10 mM NgCl2, 120 mM NaCI, 10 mM 2-mercaptoethanol) for 3 hours at 37 ° C. Nae ll-SaIG l-fragment (399 bp) is recovered in 6% PAAG.

1 μg of the plasmid pBR 322 DNA is co-hydrolyzed with restrictase EcoR I (5 units) and SaIG I (5 units) in buffer 2 for 1 hour at 37 ° C. EcoR 1-SaIG 1 fragment (3710 bp) is isolated of

0.8% agarose. A mixture of 0.25 pkmole EcoR I-SalG I-vector pBR 322, 1 pkmole Hae ll-SaIG 1-plL-2 fragment and 20 pkmole of phosphorylated oligonucleotide AATTCATGGCGC is kept in 30 μl li0 gaseous buffer (20 mM Tris-HCl, pH 7 , 5, 10 mM MgCIa, 10 mM 2-mercaptoethanol, 1 mM ATP) c10EDT4 DNA ligase for 16 hours at 10 ° C. The reaction mixture is heated for 10 minutes at 70 ° C, dNTP is added to 1 mM concentration, 5 units . DNA polymerases (Klenow fragment) and incubated for another 10 min at 10 ° C. 10 µl of the mixture is used to transform competent E. coli JM 103 cells. From colonies with the Apr, Tcs phenotype, isolate

0 plasmid DNA, designated pBIL-2, and carrying out Nar I and (EcoR l-SaIG) restriction enzymes. The correctness of the structure of nucleotides in the region of the EcoR I and SaIG I sites is confirmed by the Maxam-Gilbert method.

5P p and m e R 2. Construction of recombinant plasmid pRIL-2.

20 μg of the replicative form of the Mia rpm phage DNA is hydrolyzed in 10.0 μl of buffer 2 50 units, restriction enzyme EcoR I for 1 hour at 37 ° C. EcoR

0 l-EcoRI fragment (198 bp) was isolated in 6% PAAG.

1 µg of the plasmid pBIL-2 DNA is hydrolyzed in 20 µl of buffer 2 with restriction enzyme Iog under the conditions described above. A mixture of 0.25

5 pMol of the linear form of pBIL-2, I pkmole (EcoR l-EcoR) -Mtz fragment rpm and 10 units. T4 DNA ligases are incubated in 50 µl of ligase buffer for 16 h at 10 ° C. 10 µl of the reaction mixture is used for

0 transformation of competent cells of E. coli JM 10. Recombinant clone DNAs are analyzed by (Sma 1-HQ) hydrolysis. The primary structure of the recombinant DNA pRIL 2 is confirmed by the Maxam5 Gilbert method.

Example Construction of recombinant plasmid plL-2/21.

10 μg of pRIL-2 DNA is hydrolyzed sequentially in 50 μl of buffer 1 and buffer 2

The restriction endonucleases Pst I (20 units) and SaIG I (20 units) Pst l-SaIG l-fragment (1359 bp) were isolated by electrophoresis.

10 μg of plasmid pDSt + o are hydrolyzed jointly with Pst restriction endonucleases

5 I (10 units) and Xho I (10 units) in 50 µl of buffer I for 1 h at 37 ° C. (Pst l-Xho 1) -fragment was separated by electrophoresis in 4% PAAG. A mixture of 0.25 pcmol of the linear form pDSt2 + o, 0.25 pcmol of Pst 1-SaiG l-fragment pRIL-2, and 10 units. T4 DNA ligases in 50 µl of the ligase buffer were kept for 10 h at 10 ° C. 10 µl was used to transform competent B. coli JM 103 cells. The plasmid plL-2/21 was isolated from the colonies with the Apr phenotype and analyzed with endspinosis Bsp I and EcorI

PRI me R 4. Expression of the IL-2 gene in E coli cells SG-20050

The recombinant plasmid plL-2/21 is transformed into E. coli SG20050 cells. Bacterial cells are cultured in 5 ml of LB medium at 37 ° C to a density of 05500 0.6-0.7, then mitomycin C is added to the medium to a concentration of 1 mM and cultured for another 4 hours under the same conditions. 1 ml of the cell suspension is centrifuged and the cells are resuspended in 100 µl of lysing buffer (0.15 Mtris-HCI, pH 6.8, UmM 2-mercaptoethanol, 2% SDS, 5% glycerol, 0.1% bromophenol blue) 10 µl of the obtained lysate is applied to 15% PAAG with 1% SDS and electrophoresis is carried out. The gel plate is treated with 1 h of 10% TCA in 20% aqueous ethanol, then placed in 0.25% Coomassie R-250 solution for 30 minutes at 56 ° C, then kept in 10% acetic acid in ethanol. The content of IL-2 in the cell lysis is 30-35% of the total protein

PRI me R 5 ELISA analysis of cell lysates of the producer strain of human IL-2

Cells of strain E coli SG 20050, containing the plasmid piL-2/21, are grown in 5 ml of LB medium in the presence of 1 μm mitomycin C at 37 ° C to density. The cell suspension is centrifuged at 20 ° C for 2 min, 5000 rpm . The supernatant is removed and the cells are resuspended in 400 µl of TE buffer. Equal volumes of water and lyse buffer are added to 10 µl of the resulting cell suspension. The mixture was incubated for 3 minutes at 100 ° C, 10 µl of lysate was applied to 15% PAG with 1% SDS, and Lemley electrophoresis was performed. After electrophoresis, the gel plate was covered with a nylon filter and electrolyzed for 1 hour at a current of 70 mA. Filter transferred to a 1.5% solution of BCA and gelatin prepared on buffer 3 (25 mM Tris-HCl, pH 8.0, 150 mM NaCI, 0.05% Tween 20), and incubated for 1 hour at 20 ° C. Then add mouse monoclonal antibodies to human interleukin-2 to a concentration of 10 μg / ml are kept and incubated for 12–16 h at 20 ° С. The filter is thoroughly washed with buffer 3 and incubated and 20 ° C for 2 hours with rabbit antibodies against mouse IgG conjugated with horseradish peroxidase. The filter is washed with buffer 3 to stain the strips.

it is placed in 6 ml of 0.05% 2-chloronaphthol in 15% aqueous ethanol containing 80 mM Tris-HCl (pH 7.5) and 10 µl of 30% hydrogen peroxide is added.

PRI me R 6 Determination of the biological activity of recombinant IL-2

Cells of strain E coli SG 20050, containing the plasmid plL-2/21, are grown in 5 ml of LB medium in the presence of 1 mM mitomycin C at 37 ° C until density. The cell suspension is centrifuged at 20 ° C (2 min, 5000 r / min) The supernatant is removed, and the cells are resuspended in 2 ml of phosphate buffer and sonicated. The precipitate is collected by centrifugation and dissolved in 2 ml of 6M guanidine chloride. The solution is centrifuged (15 min., 10,000 rpm), the supernatant is dialyzed against phosphate buffer 14- 16 h at 4-6 ° C. The biological activity of the obtained lysate is determined by the method of Gil Isa content of human IL-2 in the cell lysate is (3-4), 108 IU / L

Claims (3)

Invention Formula 1 The recombinant plasmid plL-2/21 DNA encoding human interleukin-2, measuring 3,772 poi mol 2.5 Md and containing -EcoR l-EcoR I - DNA fragment of phage M13 rpm with size of 0 2 thousand p o with the regulated region of the rec A gene Proteus rmrabiiis -Pst l-Xhol - plasmid pDSt2 + o DNA fragment of 2.38 pp with transcription terminator t + o and 3-terminal part of the ampicillin resistance gene -Pst l-EcoR I DNA fragment of plasmid pBR 322 with size of 0 74 thousand along with 5-end part of gene -Haell-SalGI DNA fragment of plasmid plL-2, 0.4 thousand by with the synthetic gene of human IL-2 - genetic marker Na - ampicillin resistance gene, -unique sites Psf I Smal and Pvull 2 In a manner construct plasmid DNA recombinants tnoj plL-2/21 encoding human interleukin-2 comprising that sequentially ligated synthetic gene of human IL-2 with the artificial oligonucleotide AATTCATGGOGC regulator - the secondary region gene rec A Proteus mirabilis and then Rstl-Xhol - fragment pDSt2 + o and transformation of the obtained ligase mixture of E coli M103 cells with the subsequent selection of colonies containing the target plasmid DNA 3. The bacterial strain Eschenchia coli BKM B-5542 - producing human interleukin-2