Recombinant plasmid dna rker-9 encoding human erythropoietin, strain cultured ovary cells chinese hamster snore-9 - producing human erythropoietin

 

(57) Abstract:

The invention relates to the field of biotechnology. By embedding Bst EII - BglII fragment (2383 N. p.) plasmid pSV - Ep - gpt, including the structural gene for erythropoietin (EPO) man in the Sma I site of the vector plasmid pNut (5733 N. p.) containing activated metallothioneine the promoter of the mouse MT - I gene digidrofolatreduktazy under the control of the early promoter, SV-40, receive recombinant plasmid vector rker-9, having a size 8116 N. p. (mol. m 5 MD) and providing the biosynthesis of EPO person in cultured cells of the ovary of the Chinese hamster. As a result of cell transformation SNO-Idhfr constructed recombinant plasmid of rcer-9, subsequent cultivation on the growth environment and selection of colonies resistant to methotrexate and hybridization with DNA rker-9 obtained recombinant strain Snore-9. Cells of a new strain in stationary cultivation steadily produced into the culture fluid EPO with output 400% act./ml of medium. 2 S. p. f-crystals, 2 Il.

The invention relates to biotechnology and molecular biology, namely, genetic and cell engineering, of interest for large-scale production of recombinant natural erythropoietin is ur describes several works on gene expression of EPO in animal cells and the development of strains-producers of recombinant EPO on the basis of the cells of rodents and primates, for example on the basis of cell line Cho (cells Chinese hamster ovary).

Known plasmid pDSVL-gHuEPO containing a full-sized gene EPO person under the control of the promoter of the late genes of SV40 virus, and minigun dihydrofolate reductase (DHFR) mouse [1, 2]. After transfection expressing vector pDSVL-gHuEPO cell line CHOdhfr and selection in an environment without nucleotides derived cell clone secreting recombinant EPO in the culture liquid with a yield of 18.2 per unit of activity (%act.) on ml [3]. By conducting several rounds of amplification in the presence of increasing concentrations of methotrexate was obtained producing strains stably secreting EPO with access to 100% act./Jr.

Known plasmid pRKFL13 containing cDNA EPO gene under the control of the main late promoter of adenovirus, a polyadenylation site, enhancer and the site of initiation of replication of SV40 virus, the virus-associated gene of adenovirus, and DHFR gene under the control of the early promoter SC40 [4]. By transfection with plasmid pRKFL13 CHOdhfr-cells with subsequent selection of transfectants in the absence of nucleotides, as well as holding three rounds of amplification in an environment with methotrexate obtained producing strains of EPO secreting the hormone in combinatio EPO cells-producers, what complicates procedures for the isolation and purification of the hormone.

Known plasmid DNA pSV-sEp-poly-Neo and the strain of cultured Chinese hamster cells CHO-PE SCC(P) N 626D - producer EPO man, described in [5 prototype]. Plasmid pSV-sEp-poly-Neo obtained by cloning DstEII - BglII DNA fragment length 2386 nucleotide pairs (N. p.), containing the complete gene for EPO person in the vector pSV2 gpt [6, 7]. The building is the restriction sites HindIII - BglII, followed by deletion of the district HindIII - BstEII and merge with the plasmid pSV2 neo site enzyme Eco RI. By transfection with recombinant plasmid pSV-sEp-poly-Neo and plasmid pTK-1 ovary cells Chinese hamster CHOtk and subsequent selection of transfectants in a medium containing gipoksantin, aminopterin and thymidine (GAT), the selected cell clone CHO-PE containing in their genome integrated copies of the EPO gene and stably secreting recombinant hormone into the culture medium.

The disadvantage of this producer is that of an integrated gene erythropoietin is in the cell in a single copy and it is not possible to achieve an increased production of this protein due to the increased dose of the target gene.

An object of the invention is uprage synthesis of EPO cells-producers.

The problem is solved by using as a vector plasmids pNut [8], which used activated metallothioneine the promoter of the mouse MT-1 gene dihydrofolate reductase DHFR and design on the basis of recombinant plasmids pKEP-9. With the aim of raising the level of biological synthesis promoter close to the structural part of the full gene EPO man, just beyond the genome is the signal splicing and polyadenylation site of the early region of the SV40 virus. This combination of regulatory elements determines the high level of gene expression of EPO and efficiency of the synthesis of the recombinant hormone in animal cells. The presence of plasmids pKEP9 active gene dihydrofolate reductase DHFR under the control of the early promoter, SV-40 allows for the selection and amplification of alien sequences integrated into the host cell genome DURX-B11 (dhfr - variant cell line CHO-K1), in medium containing methotrexate. The combination of transfection, selection and amplification allows to obtain a cell line producers CHOpE-9, containing in its genome integrated copies of the gene EPO (natural form of EPO) and stably secreting recombinant hormone into the culture fluid.

Strain-produce is respecti, i.e. is resistant to high doses of methotrexate (500 nm), and synthesizes recombinant human erythropoietin with output of up to 400 units of the act./Jr.

The resulting strain is characterized by the following features:

1. Morphological features.

Culture presents epitheliopathy and spindle-shaped cells with large nuclei are irregular in shape, containing from one to three nucleoli. The granular cytoplasm, vacuolization.

2. Cultural characteristics.

Strain CHOpE-9 supported on a mixture of domestic environments Needle MEM with the addition of 10% fetal calf serum (ETS) and 1% non-essential amino acids. Cell substrate dependent, form a monolayer. Separation of cells from glass or plastic spend a mixture of solutions of 0.02% of versene (2/3) and 0.25% trypsin (1/3), the multiplicity of sieving 1:3 to 1:4, sowing dose of 100-120 thousand cells in 1 ml of a Dense monolayer culture reaches in 3 days after sowing.

3. Resistance to antimetabolites.

When culturing cells of a strain resistant to methotrexate at a concentration of 500 nm, due to the presence integrated into the genome DNA sequences.

4. Cryopreservation.

Cryoconserved the tread. Concentration of 3-5 million cells in 1 ml of Viable after the recovery remain 95% of the cells.

5. Karyological characteristics.

Karyological analysis of strain CHOpE-9 spend on drugs, cooked without burning out the release in the flame of a spirit lamp and stained with Azur-Sosina.

The frequency of polyploidy in a population of dividing cells is determined on the basis of counting 1000 cells. The proportion of polyploid cells is 8%.

Analysis of the distribution of cells according to the number of chromosomes in preparations stained with Azur-Sosina. The viewer is subjected to 50 cells. The modal class of 20 chromosomes.

Conducted featurecontrol 15 metaphase plate. It is established that the karyotype of the cells CHOpE-9 stable. The main set consists of chromosomes 1-, 2-, 4-, 5-, 7-, 9-, 10-th pair. X-chromosome rebuilt and forms a marker chromosome M3. Identified 11 recurrent marker chromosomes (Fig. 2).

6. Control of species identity.

The line of sight is confirmed by analyzing the electrophoretic mobility of isoenzymes - glucose-6-phosphate dehydrogenase and lactate dehydrogenase in polyacrylamide gel. Cells have isozyme set of characteristic cells of China the e bacteria, fungi and Mycoplasma) receive negative results at levels 10 and 20 passages.

8. The productivity of the strain.

Producing strains CHOpE-9 synthesizes recombinant human erythropoietin with the release into the culture medium up to 400% act./Jr.

The proposed recombinant plasmid pKEP-9 and the strain of cultured Chinese hamster cells CHOpE-9 for the first time obtained by the authors of the present invention, are not described in literature, therefore, we can conclude that the technical solutions according to the criteria of the invention of "novelty" and "inventive step".

List of figures graphical images

Fig. 1. Scheme design and genetic map of the recombinant plasmid pKEP9. Bright blocks labeled DNA sequence of the SV40 virus, the dark blocks - regions of the gene dihydrofolate reductase (DHFR), a thin line - DNA-fragments of the plasmids pUC18, points marked the promoter MT-1, the hatch - the remainder of the gene coding for xanthine-guanine phosphoribosyl transferase of E. coli (gpt), gray blocks - EPO gene. Arrows indicate the directions of transcription of the genes. Plasmid pKEP9 is composed of: - BstEII-BglII fragment of plasmid DNA pSV-Ep-gpt size 2383 N. p., containing the gene for erythropoietin;

-EcoRI-SalGI - fragment plasmalogen plasmid DNA pNut size 600 N. p., containing the signal splicing and polyadenylation site;

- Cloned-HindIII fragment of plasmid DNA pNut size 324 N. p., contains the website initialization of replication (ori) and the promoter of the early genes of SV40 virus;

- Cloned-SmaI/BstEII fragment of plasmid DNA pNut size 650 N. p., containing the promoter metallothionein gene in mice (MT-1);

- HindIII-SalGI - fragment of plasmid DNA pNut size 1500 N. p., containing DHFR cDNA and the polyadenylation site.

Fig. 2. The karyotype of culture. Differential G-colouring of the chromosomes of cells CHOpE-9.

The following are examples of implementation of the invention.

Example 1. Construction of recombinant plasmids pKEP-9.

As a source of genetic sequences coding for EPO man, using plasmid pSV-Ep-gpt (Fig. 1), which contains BstEII-BglII-fragment DNA EPO person length 2386 N. p. [5]. Cells of E. coli HB101 transformed with the plasmid pSV-Ep-gpt, and cells of E. coli HB101 transformed with plasmid pNut [8], is grown in one liter of LB medium [9] with ampicillin at a concentration of 50 μg/ml for 20 hours, the Cells are precipitated by centrifugation (5000 g, 5 min, 4oC) and allot of them plasmid DNA, as described in [10].

Received preparadigm way. Carry out the hydrolysis of 60 µg DNA pSV-Ep-gpt restriction endonucleases BstEII and BglII (step 1 in Fig. 1) in buffer containing 0.01 M Tris-Hcl (pH 8.0); 0.01 M MgCl2, of 0.05 M NaCl, 1 mM-mercaptoethanol. Similarly conduct hydrolysis of 20 μg DNA pNut by restriction enzyme SmaI (step 2 in Fig. 1). Hydrolysates plasmids subjected to electrophoresis in 4% SDS page, gel bromide stain with ethidium under long-wave ultraviolet irradiation cut a strip corresponding to linear DNA, for SmaI digests pNut and the band corresponding to fragment 2383 N. p., BstEII-BglII digests pSV-Ep-gpt. DNA fragments isolated from the gel pieces by Electrosila on paper disks DE-81 (Whatman, USA) in 0.01 M Tris-borate buffer, pH 8,3, at 350 V for 2-3 h DNA disks wash 1.5 M NaCl; 0.01 M Tris-HCl (pH 7.5); 1 mm EDTA (420 μl) for 40 sec at 56oC, precipitated with ethanol. Sediments washed with alcohol, dried, dissolved in 10 μl of water. The protruding 5' ends of the selection BstEII-BglII complete to blunt during a polymerase reaction using fragment maple DNA polymerase I of E. coli [11]. After completion of 1 pmol fragment is mixed with 0.2-Ptolemy SmaI-linearized and dephosphorylated vector DNA pNut in 30 μl of a solution containing 66 mm Tris-Hcl (pH 7.5); 5 mm MgCl2; 5mm dithiotreitol (DTT; 1 mm ATP; 14 units of the act. DNA ligase fpedantic cells of E. coli HB101, as described in [12] . Transformed cells are plated on plates with solid agar containing ampicillin (50 μg/ml), and cultivated for 18 h at 37oC. the Grown colonies break in 5 ml of LB medium containing ampicillin (50 μg/ml), incubated at 37oC and intensive shaking for 18 hours, the Cells are separated by centrifugation of them produce plasmid DNA for subsequent restriction analysis. The resulting plasmid is called pKEP-9 (Fig. 1).

Example 2. Getting a producer strain CHOpE-9.

To obtain a stable producer recombinant EPO person conducting the transformation ovary cells Chinese hamster CHO-K1 dhfr - plasmid pKEP-9. Cells cultured in medium F-12 (90%), with the addition of 10% fetal calf serum (ETS). In culture flasks (25 cm2seeded with 5 ml of cell suspension (1.5 million cells) and after 16-20 h standard method of calcium-phosphate precipitation conduct transformation [13]. Plasmid DNA added to the cells in the form of calcium-phosphate precipitate. The precipitate prepared as follows: at room temperature in a bottle make A 30 µg DNA plasmids in 300 µl of TE buffer (1mm Tris-HCl; 0.1 mm EDTA; pH 9,0) add 188 μl 2M CaCl2and water to 1500 ml. In the presentations to the contents of vial B and leave for 30-40 min at room temperature before the formation of translucent precipitate. Vials with cell culture CHO-K1 dhfr - drained medium, on the monolayer put 3 ml of precipitate and incubated for 45-60 min at 37oC, then add 10 ml of medium F-12 containing 2% ETS. After 16 h after transformation, cells are washed and placed in growth medium for 24 h Later cells removed from the vial with a mixture of trypsin-versene and scatter at a concentration of 100 cells per ml on two Petri dishes with a diameter of 90 mm in selective medium (a-MEM (without thymidine and gipoksantina) containing 10% ETS and 500 nm methotrexate. Cells incubated in selective medium for two weeks, a change of environment carried out every three days. 14 days after placing in the selective conditions are formed colonies resistant to methotrexate. Colonies emit, scatter in the wells of 48-hole plastic tablets in the environment of a-MEM containing 10% ETS and 500 nm methotrexate and incubated at temperature 37oC, 98% humidity in an atmosphere of 5% CO2before the formation of the monolayer. The hybridization define cells which clones contain in their genome a DNA plasmid pKEP-9 [14].

The presence of EPO in the culture fluid is determined by the inclusion of in vitro tritium-labeled thymidine growing in splenocytes of mice with phenylhydrazine anemia [15] . The analysis is performed clebert day. On the third day after the last injection sterile extract of spleen and preparing a suspension of splenocytes at a concentration of 4 million cells/ml in RPMI medium 1640 containing 20% ETS. In the wells of flat-bottomed 96-well tablet make 50 ál of cell suspension, add an equal volume of dilutions of the test material in the culture medium. As a positive control, use a dilution of a standard sample of EPO with an activity of 250 units of the act./ml production Boehringer Mannheim (Germany). As a negative standard serves as a cultural medium. Tablet incubated for 22 h at 37oC in a humid atmosphere containing 5% CO2. Next to each hole making 1 µci of [3H]-thymidine in 20 µl of RPMI medium 1640. Incubation continued under the same conditions for 2 hours the Contents of the wells is transferred onto glass fiber filters GF/C (Whatman, USA) using a harvester Titertek (Flow, USA), washed several times with water. The filters are dried in air and calculate in toluene scintillator counter radioactivity MarkIII (Beckman, USA). The biological activity of the tested samples are estimated by comparison with a calibration curve EPO-standard.

Thus, the selected clone CHOpE-9 cells, which stably prod is verovanii.

Example 3. Isolation and characterization of recombinant erythropoietin.

The culture fluid after growing cells-producers CHOpE-9 in volume of 3000 ml filtered through filter paper, make a coherent concentration to a volume of 50-60 ml and diafiltration against 500-600 ml 15 mm Tris-HCl, pH 7.0, containing 0.2 mm of copper sulfate and 0.02% tween-20 (buffer A) using ultrafiltration separation apparatus for hollow fibers AR-0.2 n production Bureau BLS, Kirishi. The resulting concentrate of the culture fluid is applied on the column with 50 ml DEAE-sepharose, balanced with buffer B (15 mm Tris-HCl, pH 7.0, 0.02% tween-20). The column is washed successively with 50 ml buffer A; 150 ml of 6M urea, acidified with acetic acid to pH 4.5; 100 ml 25 mm NaCl in buffer; 100 ml 80 mm NaCl in buffer; 100 ml 160 mm NaCl in buffer at a speed of 4 ml/min Fractions of the eluate are collected by the optical density determined at a wavelength of 254 nm using a flow-through UV densitometer and determine their activity enzyme-linked immunosorbent assay (ELISA) using EPO-specific polyclonal rabbit antisera and conjugates antivitamin antibodies (goat-anticolic) with horseradish peroxidase. the tym (pore diameter of 30 nm) sorbent, containing butylene group, equilibrated with 5% solution of acetonitrile in 0.1% triperoxonane acid. The elution of proteins with speakers spend a linear gradient of 5-90% acetonitrile over 60 min at a speed of 5 ml/min Fractions collected, examined for the presence of EPO activity, as described above, are combined and concentrated three times by evaporation on a rotary evaporator Buchi (Switzerland). The sample obtained rechromatography the above column with obremenitve sorbent. Faction, eluruumina with columns 48-55% acetonitrile and having EPO activity are pooled, evaporated on a rotary evaporator at 30oC, dissolved in 0.02 M sodium citrate, pH of 6.9, containing 0.1 M NaCl. Samples of recombinant erythropoietin are characterized by the following indicators:

1. The purity and homogeneity.

the homogeneity of the preparation is not less than 90-95% (according to gel electrophoresis in a 13% polyacrylamide gel densitometry);

the relation OP280/OP260= 1,05;

content aggregated form to 4.5% (according to the Western blot turns and densitometry of electrophoregram, painted silver);

the impurity content of the DNA of the cells-producers of not more than 10 PG per 10,000 units of activity ( CLASS="ptx2">

2. Identity.

- molecular mass - 37500 Yes (according to gel electrophoresis with protein markers of molecular weights);

- specific activity to 150,000 IU/mg (10000 u/ml) (determined by the method of solid-phase ELISA);

- specific activity in vitro 200000 IU/mg (determined by incorporation of [3H] -thymidine growing in splenocytes of mice with phenylhydrazine anemia [15]);

- specific activity in vivo is not less than 100,000 IU/mg (determined by counting the number of reticulocytes in blood samples of mice after introduction of samples of EPO on [16]);

- the amino acid composition corresponds to the calculated data (determined after hydrolysis of 6 N. HCl, 110oC, 24 h);

N-terminal sequence of A-P-P-R-L-I-C-D-S-R 10 amino acids corresponds to the literature data (determined by the method of Edman on automatic sequencing machine with the identification of FGF-amino acids by HPLC).

References

1. Lin, F. K., Suggs, S., Lin C. H., J. K. Browne, Smalling R, Egrie, J. C. Chen, K. K. , Fox, G. M. , Martin F., Stabinsky, Z., Badrawi S. M., Lai, P. H., Goldwasser E. Proc.Natl.Acad.Sci.USA. 1985. V. 82, N 22. P. 7580-7584.

2. U.S. patent N 4703008. The DNA sequence encoding erythropoietin.

3. Urlaub, G., Chasin L. A. Proc.Natl.Acad.Sci.USA. 1980. V. 77, N 7. P. 4216-4220.

4. The international application N 86/03520. The way Bodiroga human erythropoietin, strain cultured cells, Chinese hamster ovary CHO-PE - producing human erythropoietin. The decision to grant a patent from 28.03.95. (RF patent N 2070931, bull.From. 9636, 27.12.96).

6. Mulligan R. C., Berg P. Proc.Natl.Acad.Sci.USA. 1981. V. 48, N 4. P. 2072-2076.

7. Richardson K. K., J. Fostel, Skopek, T. R. Nucl.Acids Res. 1983. V. 11, N 24. P. 8809-8816.

8. Palmiter R. D., Behringer, R. R., C. J. Quaife, F. Maxwell, Maxwell I. F., R. L. Brinster, (1987) Cell 50, P. 435-443.

9. Miller, J. Experiments in molecular genetics. TRANS. from English. - M.: Mir, 1976, S. 395.

10. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering: Molecular cloning. TRANS. from English. - M.: Mir, 1984, S. 98-103.

11. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering: Molecular cloning. TRANS. from English. - M.: Mir. 1984. S. 123-124.

12. Maniatis T., Fritsch E., Sambrook J. Methods of genetic engineering: Molecular cloning. TRANS. from English. - M.: Mir. 1984. S. 240-241.

13. Gorman, K. Highly efficient gene transfer into mammalian cells. In kN. : DNA cloning. Methods. Ed. by D. Glover Lane. from English. - M.: Mir. 1988. C. 409-463.

14. Rothstein R. Cloning in yeast. In the book: DNA cloning. Methods. Ed. by D. Glover Lane. from English. - M.: Mir. 1988. S. 299.

15. Krystal G. Exp.Hematol. 1983. V. 11, N 7. P. 649-660.

16. Gold person, size 8116 nucleotide pairs and molecular weight of 5 Mg, containing Bst EII - Bgl II fragment of plasmid DNA pSV-Ep-gpt, size 2383 N. p.; SmaI - SmaI fragment of plasmid DNA pNut, size 5733 N. p.; unique restriction sites: E coRI, Sal GI; genes and genetic markers: bla gene for synthesis-lactamase gene DH FR for synthesis digidrofolatreduktazy, erythropoietin gene for synthesis of recombinant human erythropoietin.

2. Strain cultured ovary cells Chinese hamster Snore-9 - producing human erythropoietin.

 

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