Synthetic dna encoding human erythropoietin, comprising its vector, method of production of erythropoietin producer strain, erythropoietin producer strain

FIELD: biotechnology.

SUBSTANCE: synthetic DNA is proposed, encoding human erythropoietin, having the sequence Seq ID No. 1, comprising its expression vector, the method of production of erythropoietin producer strain, and a strain of a Chinese hamster ovary cells - producer of recombinant human erythropoietin, deposited under the number RKKK(P) 761 D.

EFFECT: invention enables to increase the expression level of recombinant human erythropoietin.

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Field of the invention

This invention relates to the field of medicine and biotechnology.

The level of technology

Erythropoiesis, the formation of red blood cells, is continuous throughout a person's life to compensate for destroyed cells. Erythropoiesis is a highly regulated physiological mechanism that allows it to produce sufficient red blood cells for proper oxygenation of tissues. It is known that the formation of red blood cells occurs in the bone marrow under the control of a hormone protein nature, erythropoietin.

The human erythropoietin is a glycoprotein with a molecular weight of approximately 34000 Yes, which is produced by many types of tissues, but mainly by the kidneys. Polypeptide chain of human erythropoietin contains 192 amino acid residues and can be N-glycosylated at three sites. The degree of glycosylation depend on the activity and stability of the molecule of erythropoietin in vivo. The function of EPO is to stimulate the conversion of primary preceding cells in the bone marrow in proerythroblast that, maturing and becoming Mature red blood cells are carriers of oxygen. Under normal conditions, when the healthy tissue of the body receive enough oxygen, erythropoietin contained in plasmas� blood in very low concentrations (10-15 mIU/ml). This low concentration is sufficient to maintain the reproduction of red blood cells to replace retiring during aging.

However, the concentration of erythropoietin in the blood increases dramatically under conditions of hypoxia, a reduction in the transport of oxygen in the blood. Hypoxia can be caused, for example, the loss of large amounts of blood during hemorrhage, destruction of red blood cells due to radiation, reduction in oxygen consumption when in high layers of the atmosphere, as well as with anemia (state S. A., Simbirtsev A. S. cytokines, St. Petersburg, 2008). In particular, the sharp drop in the concentration of erythropoietin in the blood in patients with chronic renal insufficiency, which, in turn, causes the characteristic of the condition of anemia.

All of the above explains the widespread clinical use of erythropoietin, in particular recombinant human erythropoietin for the treatment of several pathological conditions. The main result of the use of erythropoietin is associated with the ability to restore its own blood and to compensate for anemia without blood transfusion. Against this background, the patients with significantly improved functioning of the cardiovascular system, increases efficiency and improves overall quality of life.

Rekom�inantly human erythropoietin receive clearance from the environment of the cultivation of cells of higher eukaryotes, transformed with genetic constructs for synthesis of erythropoietin. The process of obtaining cell lines (strains cells) - producers involves a long process of selection of such strains with multiple selection of the most productive and stable production of erythropoietin cells, followed by the adaptation of selected clones to grow in the serum-free cell culture medium. Purification of erythropoietin from the environment of cell culture usually includes several chromatographic steps. Due to the constant expansion of production of this protein for its use in medical practice and high cost of production constantly working to improve the productivity of cell lines producing erythropoietin and optimization of cultivation conditions for the cells.

The closest to the present invention is a clone of Chinese hamster ovary cell IN producing human erythropoietin (EE). Cells of this clone under cultivation in the conditions of adhesion of growth in serum-free medium for 7 days were allocated to the environment 91 mg erythropoietin/L.

Summary of the invention

The problem solved by the authors, was to achieve a high level of cells to produce recombinant human erythropoietin when they are cultured in Bassington�th medium with chemically defined composition.

The technical challenge was constructing expression vectors, obtaining transfected these vectors Chinese hamster ovary cell, selection of the most productive and stable for this trait clone of cells with the subsequent study of the dynamics of cell growth and accumulation of the target product - recombinant human erythropoietin, the analysis of the biological activity of recombinant erythropoietin in comparison with the reference sample.

This problem is solved by the fact that the proposed synthetic DNA encoding human erythropoietin having the sequence Seq ID No. 1. Specified DNA is intended for expression in cells of the Chinese hamster ovary.

Also provided an expression vector containing this DNA.

A method of obtaining strain-producer of erythropoietin, which includes the transfection of cells defective in dihydrofolate reductase gene, a mixture of two expression vectors containing the DNA according to the invention is one which contains as a selective marker gene of resistance to neomycin/G418, and the second contains as a selective marker gene for resistance to methotrexate, followed by selection of clones, both resistant to G-418 and methotrexate.

Advantageously, when in this method, the cells are cells of the ovary Ki�ayskogo hamster.

We have also proposed a strain of Chinese hamster ovary cell - producer of recombinant human erythropoietin, deposited under number RCCC(P) 761 D.

Detailed description of the invention

The technical result was achieved using a high performance and stable strain of Chinese hamster cells (Cho) - producer of recombinant human erythropoietin, which provides accumulation in the culture medium with 300 mg/l erythropoietin at 12 days of cultivation in serum-free medium with chemically defined composition.

This strain of Chinese hamster ovary cell Cho-EPO/A - producer of erythropoietin person was placed in a Specialized collection of cell cultures vertebrate Russian cell culture collection 05.09.2013 No. RCCC(P) 761 D.

Strain is a primary producer of erythropoietin.

Pedigree strain:

The parent cell line CHOdhfr-. Transfection of plasmids pBVneo/hER and pBVdhfr/hER selection stable high-yielding clone in the medium without hypoxanthine/thymidine with 500 μg/ml G-418.

The number of passages by the time of the Deposit - 15.

Standard growing conditions: Medium CD CHO (hivitrogen) without hypoxanthine/thymidine with 8 mm glutamine, 500 µg/ml G-418, 37°C, 8% CO2.

Cultural properties: Suspension cultivation in test tubes, black�Oh or 6-hole circuit boards on an orbital shaker. Sowing dose of 0.2-0.3×106/ml, subculture every 3-4 days. Doubling time 22-24 hours.

Data on species: Cricetulus griseus (PCR analysis with species-specific primers).

Produces recombinant eritropoyetina person (estimated using enzyme-linked immunosorbent assay, Western blot, biological test).

Recombinant human erythropoietin secreted into the culture medium at a concentration of not less than 8 PG per cell per day. The stability of the cultivation of at least 25 passages. The definition of products using enzyme-linked immunosorbent assay.

Method of cryopreservation: fetal serum with 10% DMSO, freezing to -70°C at a rate of 1°C/min, then the rooms in liquid nitrogen, the viability after thawing and washing of cryoconserved 85% (with trypan blue).

Perhaps culturing cells in DMEM with 10% fetal serum (adhesive growth) without additional adaptation.

Graphic materials

Fig. 1 - map of plasmid vector pBVneo/hER.

Fig. 2 - map of plasmid vector pBVdhfr/hER.

Fig. 3 - Dynamics VCD cells seven candidate clones.

Fig. 4 - Dynamics of cell viability seven candidate clones.

Fig. 5 - Dynamics of production of repo cells seven candidate clones.

Fig. 6 - Dynamics VCD, viability and level of repo in cell culture clone E feathers on�'s passages passage.

Fig. 7 - Dynamics VCD, viability and level of repo in cell culture clone E after 2 months of continuous passaging.

Fig. 8 - cell Proliferation of TF-1 under repo extracted from the supernatant of cells of strain E, sample and reference TIME.

Seq ID No 1 sequence artificial gene encoding human erythropoietin.

The invention is illustrated by the following examples:

Example 1. The generation of vector constructs for expression of erythropoietin in Cho cells

The design of the nucleotide sequence of the gene encoding human erythropoietin, was carried out taking into account the frequency of use of codons in the genome of Chinese hamster. The resulting sequence is represented in Seq ID No. 1 encodes the sequence of human erythropoietin, including the signal peptide, and flanked with 5'-end a restriction site BamHI and Kozak sequence, and 3'-end - additional stop codon taa, located directly after the "natural" stop codon tga, and the restriction site Xbal. This sequence was synthesized by automated chemical synthesis and pasted the above restriction sites in two expression vector, the first of which contained as a selective marker gene of resistance to neomycin/G-418, and the second gene is dihydrofolate reductase.The result is two expression plasmids, pBVneo/hER and pBVdhfr/hER, respectively, of the map which is shown in Fig. 1 and 2. The accuracy of gene synthesis and Assembly of plasmids were confirmed by sekvenirovanie.

Example 2. Transfection of cells and selection of highly productive strains

For transfection was used the cell line CHO-S(Dhfr -) adapted to suspension growth in serum-free medium. Cells were cultured in the medium CD-CHO Medium (Invitrogen) with the addition of 8 TM Ultraglutamine (Lonza) and HT media supplement (Invitrogen). Transfection was performed using the reagent Lipofectamine™ 2000 (Invitrogen). For transfection used a mixture of plasmids pBVneo/hER and pBVdhfr/hER in a ratio of 1:1. Selection was performed in medium CD-CHO Medium (Gibco) with 8 mM Ultraglutamine (Lonza) and 500 μg/ml of antibiotic G-418 (Gibco).

48 hours after transfection the cells were transferred to selective medium, which was cultured for 25 days, and then cloned by the method of limiting dilutions. Was analyzed 240 individual clones that demonstrated stable growth in the selective medium. Individual clones were identified visually, with increasing number of cells were removed at 24-hole boards, where were collected the supernatants for primary screening, which was performed using ELISA (Human Erythropoietin Platinum ELISA, eBiosciences). Just after primary screening were selected for further work 50 clones in the supernatants which the level of repo PR�visil 2 μg/ml,

Cells 50 clones with maximum production according to the results of primary screening were expanded and seeded at the density of 3×105/ml in 1 ml culture medium in the wells of a 24-hole boards and were cultured for 7 days. After you have determined the concentration of repo in the supernatants using ELISA.

In the second stage of screening, we selected 7 individual clones, in which the concentration of supernatants of rape was maximum. The names of these clones, as well as the concentration of repo in their supernatants when the first and second screening are shown in table 1. In subsequent experiments have been conducted more detailed study of cultural properties and productivity of seven candidate clones.

In the third stage of screening carried out the cell culture 7 clones in Erlenmeyer flasks of 250 ml, with initial density of 300 thousand cells/ml, 100 ml per vial. The medium for culturing a medium CD Cho with 8 mm Ultraglutamine and 30% nutritional supplements SSS CD EfficientFeed™ A (Invitrogen). The cultivation was performed at 37°C, 8% CO2and shaking speed of 125 rpm. Daily selected sample of each culture was measured, the density of viable cells (VCD), viability, and concentration of repo in the culture medium using ELISA. The results are shown in Fig. 3-5.

From Fig. 3- all seven candidate clones showed similar growth, reaching a peak cell density of the order of 8-10×106/ml for about 9 days, and then maintained their viability over 80% for another 4-7 days. While differences in the level of production of repo candidate clones were quite pronounced: the maximum production of up to 180 μg/ml, was recorded in clone 7F6 and V, whereas the lowest level of repo was observed in the supernatant of clone 10D10 (100 µg/ml). In the cultures of five of the seven candidate clones were marked by a sharp drop in levels of recombinant erythropoietin after 13 days of cultivation, which coincided with a significant decrease in viability of cultures. In two clones, A 5D3 and the drop in viability occurred on 15-16 day and in the supernatants for up to 15 days inclusive) concentration repo not decreased. Considering the analyzed cultural indicators, as well as the stability of repo in the supernatants, as the target of the producer strain was selected clone A.

To increase the productivity of the clone, two rounds of amplification in ascending concentrations of methotrexate (20 nm and 100 nm). Next, the culture was reklamirovali in the medium without methotrexate and cryopreserved (created by master-Bank).

Example 3. An analysis of products of repo strain-producer E

For �analysis stability of production of erythropoietin ciampoli with cells of clone A was thawed and cells were cultured in the medium CD-CHO with the addition of 8 mm glutamine, and 500 μg/ml G-418. The cultivation was performed in Erlenmeyer flasks on a shaker (working volume of 30 ml) by re-seeding 2-3 times a week for 2 months. The growth curve analysis and production of rape was conducted at the beginning of the experiment and after 2 months of cultivation.

Fig. 6 presents the dynamics of the content of living cells in culture (VCD), cell viability and concentration of repo in the supernatant of cells E in the first passages. The culture was maintained high viability for up to 14 days, the concentration of viable cells was reached within 8 days of 8×106cells/ml, and the level of repo already the 10 day was about 300 µg/ml and remained approximately at this level for another 5 days.

When passaging cells clone A within 2 months of the dynamics of the increase of cell mass after subculturing was quite stable: for 3 days the cells with 200 thousand/ml reached a density of 1.2-1.4 million/ml Viability under this ranged from 88% to 97%. After 2 months of passaging re-evaluated the growth and production of repo. The results are presented in Fig. 7. As can be seen from the figure, the dynamics of cell growth was virtually the same as at the beginning of cultivation - the maximum density was achieved on the 9th day (about 10 million/ml), high viability was maintained for 12-13 days, and the plateau level of rape in the supernatants was achieved on day 10-11. Peak concentration� target protein was 320 μg/ml.

Thus, continuous cultivation for 2 months highly productive cells of strain A were not accompanied by significant changes in the dynamics of growth and production of target protein. This fact indicates the suitability of the cells of the strain E for industrial cultivation, including in the conditions of perfusion cultivation.

Example 4. Analysis of biological activity in the repo, produced by cells of the strain E

To study the biological activity of the recombinant protein secreted by cells of the strain E, these cells were cultured in Erlenmeyer flasks for 12 days, after which the supernatants were clarified by centrifugation at 1000 g followed by filtration through a filter with pore diameter of 0.22 μm. Purification of recombinant erythropoietin was performed using immunoaffinity chromatography (used immunosorbent column 6-A-sepharose ("Drops") volume 15 ml). The protein concentration in the obtained sample was determined spectrophotometrically, and then analyzed the biological activity in the test of proliferation of the cell line TF-1 in comparison with the reference sample of erythropoietin British Pharmacopoeia (TIME, 32500 Units in a vial). The intensity of proliferation was determined by incorporation of3H-thymidine into acid-insoluble precipitate. The results presented in Fig. 8 shows�t, what repo extracted from the supernatant of cells E, and control sample have identical TIME activity, dose-dependent enhancing cell proliferation of TF-1 in the concentration range from 0.1 to 100 μg/ml.

1. Synthetic DNA encoding human erythropoietin having the sequence Seq ID No. 1.

2. DNA according to claim 1 for expression in cells of the Chinese hamster ovary.

3. The expression vector containing the DNA according to claim 1.

4. A method of producing cell strain of Chinese hamster ovary - producer of recombinant erythropoietin, including cell transfection Chinese hamster ovary defective in dihydrofolate reductase gene, a mixture of two expression vectors containing the DNA according to claim 1, one of which contains as a selective marker gene of resistance to neomycin/G418, and the second contains as a selective marker gene for resistance to methotrexate, followed by selection of clones, both resistant to G-418 and methotrexate.

5. Strain of Chinese hamster ovary cell - producer of recombinant human erythropoietin obtained by the method according to claim 4 and deposited under the number RCCC(P) 761 D.



 

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