Recombinant plasmid dna pbk415 coding polypeptide of recombinant tissular activator of plasminogen of human being, cell line cricetulus griseus cho 1f8-producer of recombinant tissular activator of plasminogen of human being, and method for obtaining and separation of polypeptide having activity of tissular activator of plasminogen

FIELD: biotechnologies.

SUBSTANCE: recombinant plasmid DNA pBK415 coding polypeptide with sequence of tissular activator of human plasminogen, also including MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transmission enhancer CMV, internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase providing stability to geneticin (Neo) and a cassette for expression in bacteria cells of gene of β-lactamase providing stability to ampicillin, cells of line Cricetulus griseus CHO DHFR(-) are obtained so that there produced is cell line Cricetulus griseus CHO 1F8 producing recombinant protein of tissular activator of plasminogen with highly stable yield at the level of up to 190 mg/l. Cultivation of cells-producers is performed under perfusion conditions in presence of a mixture consisting of additive CHO Bioreactor supplement and sodium butyrate or dimethylsulphoxide with further separation of a target product.

EFFECT: improvement of the method.

5 cl, 5 dwg, 3 tbl, 8 ex

 

The technical field

The invention relates to biotechnology, in particular genetic engineering, and can be used to produce recombinant tissue plasminogen activator person (rtPA). The invention is a recombinant plasmid DNA RVC encoding the polypeptide sequence of recombinant tissue plasminogen activator (rtPA), and producer based cell line Cricetulus griseus CHO 1F8 containing the indicated plasmid DNA synthesizing recombinant tissue plasminogen activator.

Prior art

Tissue plasminogen activator (tPA) is a protein consisting of 527 amino acids, with a weight of about 72 kDa. He has the ability to cause catalytic degradation of fibrin (the main component of blood clots), and is used in the treatment of acute myocardial infarction, pulmonary embolism, thrombosis of the veins. Acting as a serine protease tPA binds to fibrin, acquires proteolytic activity and promotes the conversion of plasminogen to plasmin, resulting in lysis of the thrombus and reperfusion blood flow (Cell Mol Life Sci. 2011 Mar;68(5):785-801. Epub 2010 Dec 7). The unique property of tPA is its extremely high selectivity in respect associated with fibrin plasminogen, which ensures its preferential activation on the surface and fibrin clot, but not in systemic blood flow.

Compared with thrombolytic agents from the previous generation, such as urokinase and streptokinase, tissue plasminogen activators operate more efficiently. This is reflected in the fact that the latter is directly linked with fibrin and thus are directed at the primary target - the clot, unlike streptokinase and urokinase, which are less specific and activate in addition to plasminogen bound to fibrin clot, and free molecules of plasminogen (Baillieres Clin Haematol. 1995 Jun;8(2):277-90). In addition, tPA has a great time living in the body of the patient.

The natural sequence of tissue plasminogen activator was identified in 1982, Nature, 1983, Jan 20; 301(5897):214-21). On its basis was established drug of alteplase (trade name "Actilyse"), containing protein, is entirely consistent with human tPA, as well as several mutant forms tPA: tenecteplase (trade name "Metalize", differs from the natural protein replacement of three amino acids) and reteplase (trade name "Retavase", characterized by a deletion of three of the five domains of tissue plasminogen activator). Numerous studies ("thrombolytic therapy of myocardial infarction", cancer, Vol. 6, No. 3, 1998; Am Heart J. 2004 Jun; 147(6):993-8 and others) showed that the efficiency of these preparato is quite similar. The most optimal security profile has alteplase. Reteplase slightly inferior to alteplase efficiency (especially if the delay of the introduction of the drug is more than 4 hours after the first symptoms of myocardial infarction). If tenecteplase there is an increased risk of intracranial hemorrhage (A. Kulikov. Pharmacotherapy of acute coronary syndromes (ii), "Formindex Practices" issue 9, 2005, p.1-16). In addition, the last two products contain a mutant form of the protein, which can provoke an immune response in a patient, which is absent in the case of alteplase.

Currently myocardial infarction is responsible for approximately 9% of deaths in developed countries. Only in 1990, according to the who, 10912 patients from myocardial infarction were killed 1001 people, which amounted to 9.2% (Alpert, J., Francis, the Treatment of myocardial infarction. Practical guide. TRANS. from English. M., 1994. S). Unfortunately, in Russia the high cost of recombinant plasminogen activators serves as a deterrent to their widespread use. Therefore, the establishment of effective producer tPA, as well as developing a method for its receipt of the domestic production of the drug is extremely urgent and important task, which will expand the Arsenal of domestic drug among the STW for treatment of acute myocardial infarction.

Eurasian patent EA reveals a system of tPA expression in prokaryotic cells. For this purpose tPA gene inserted into a vector containing the CMV promoter, the sequence of the signal peptide of Otra and gene III protein (gp), and tPA functionally associated with the sequence Shown against the course of transcription, and in the course of transcription sequence gpIII gene. The authors indicate that the presence of a signal peptide allows the protein to acquire the correct conformation to exit the cells, and the efficiency of cleavage of the signal peptide is very high. However, it is widely known that the expression of proteins in mammalian (including human) associated with glycosylation, which cannot be implemented in the bacterial cells.

A new expression vector for tPA are available in ER. Vector pCIHtPA contains sequences tPA and DHFR (digidrofolatreduktazy) under control of the CMV-promoter, and the gene sequence SVE. Vector pCIStPA additionally contains a plot of the SV40 polyadenylation, which allowed to increase the level of expression of approximately three times compared with the vector pCIHtPA. The publication also compares the levels of protein expression in cells Cho and 293 cells, transfected with the corresponding vectors: vector pCIHtPA managed to reach values of 15 and 1300 ng/ml for vector pCIStPA - 55 and 3000 ng/ml, respectively./p>

System of tPA expression in mammalian cells (Cho) are also thoroughly described in several patents company Genentech (for example, see EP 093619, EP 0117059, US 5728565, US 5849574, US 6284247). They all reveal a vector containing the sequence of the tPA and the DHFR gene, which allows further by DHFR gene amplification, to cause amplification associated gene sequence tPA.

A similar expression system of natural tPA in cells SNO, deficient in DHFR, which is the closest analogue of the present invention described in patent RU 2046827. As in the previous cases, the authors clone sequence tPA collected from three fragments of different lengths in plasmid pBR322, optionally containing the DHFR gene. Then the obtained plasmid transformed cells SNO and conduct a series of sequential amplification using methotrexate. As a result, the output of the secreted product reaches values up to 50 PG/cell/day. The disadvantage of the used system is functionally independent of the expression of the gene for DHFR and tPA through the use of two separate SV40-promoter for each of the synthesized proteins. Initially, the high outputs of the transgene observed at used concentrations of methotrexate (up to 10 µm), usually associated with a significant level of chromosomal instability producer strain and the ri the absence of selective agent, which is very toxic and therefore not used in industrial pharmaceutical production inevitably leads to a significant decline in production during prolonged cultivation of cell lines. Accordingly, the use of this approach makes it impossible to use this method in production of therapeutic recombinant proteins.

In this context it is important to note that none of the above patents are not given data on the stability of the expression system (the number of passages, generations), which is one of the most important factors for the industrial production of protein, along with high levels of expression.

Disclosure of inventions

The present invention solves the problem of constructing plasmid DNA containing the sequence of tissue plasminogen activator (tPA), and create a cell line that allows you to receive recombinant tissue plasminogen activator (rtPA) with high output at a constant level up to 190 mg/l and maintain its performance for a large number of passages (at least 50), which is a critical parameter of the possibility of application of the producer strain for industrial production of protein.

The problem is solved by constructing recombinant plasmid DNA RVC consisting of 11383 BP,and cell line Cricetulus griseus CHO 1F8.

The introduction of various genetic elements in eukaryotic genome in the composition of the recombinant constructs used for subsequent transfection of mammalian cells, can have a positive impact on the stability of the transgene in the genome of the cells of the recipient, significantly improving the growing characteristics of culture (S.C. Makrides Gene Transfer and Expression in Mammalian Cells, (2003) Elsevier Science, chapters 10-12). Such elements, in particular MAR, are used to create industrial strains-producers, for which the critical parameter that determines the possibility of its use in production, stability is the level of production and the growth rate of the culture.

Elements attach to the nuclear matrix MAR (Matrix Attachment Regions) represent fragments of DNA in eukaryotes length of 300 to 3000 BP and play a key role in nuclear organization and chromosome architecture. Interfaze they fix the chromatin protein that forms a nuclear matrix, dividing the genome of eukaryotes independent chromatin loops (Mirkovitch, J., Mirault, M.E., and Laemmli, U.K. (1984) Cell 39, 223-232.). Thanks colocalization MAR-elements with sites of active transcription and regulatory elements in the genome, the first positively affect the level of transcription of the transgene, attracting activators of transcription and controlling the accessibility of DNA in chromatin structure. In addition to these properties MAR-elements, e.g. the MAR gene lysozyme birds, can prevent a gradual reduction in the activity of the transgenic promoter, which significantly improves the stability of the level of transgene expression (Alien, G.C., Spiker, S., and Thompson, W.F. (2000) Plant Mol. Biol. 43, 361-376).

In the process of constructing plasmids, we found that the use of MAR plasmid containing the gene rtPA, allows to maintain the level of expression of this protein with the highest stability. The advantages of the present invention also consist in the use of chemical-enzymatic method for the synthesis of the gene rtPA, which allows you to choose the optimal colony composition of a gene, to avoid the presence in the sequence of the DNA structures that impede the process of translation of cryptic splicing sites, polyadenylation. Optimization of the primary structure of the gene and the use of a transcription enhancer of cytomegalovirus CMV, and DNA fragment that provides attachment to the nuclear matrix MAR, can significantly increase the level of secretion of rtPA in a nutrient medium and, accordingly, the final yield of the target product.

Recombinant plasmid DNA RVC contains the following sequence: MAR - district attachment to the nuclear matrix gene lysozyme birds, promoter of the CMV virus, enhancer of transcription of the virus CMV, cDNA gene rtPA, internal site of translation initiation IRES, DHFR gene, polyadenylation signal VIR is sa SV40, cassette expressing the gene aminoglycoside-3'-phosphotransferase, providing resistance strain cells-producer to geneticin (Neo), and cassettes for expression in bacterial cells gene P-lactamase, providing resistance to ampicillin. Plasmid RVC (figure 4)encoding a polypeptide rtPA, characterized by the following features:

- consists of 11383 BP,

- has a molecular mass of 7.4 MDA,

- encodes the polypeptide of tissue plasminogen activator,

- ensures the stability of bacterial cells transformed with this plasmid, ampicillin and mammalian cells, transfected with the indicated plasmid, geneticin,

- contains a unique recognition sites for the following restriction endonucleases:

AhdI (7352 BP), AsiSI (508 BP), BglII (2649 BP), BipI (2079 BP), BmtI (654 BP), BssHII (5778 BP), BstBI (6062 BP), BstEII (2388 BP), Bsu36I (2245 BP), EcoRI (10035 n.o.), EcoRV (10969 n.o.), FspAI (2020 n.o.), MluI (2433 n.o.), MreI (2220 n.o.), NheI (654 n.o.), NotI (3918 n.o.), PspXI (3342 n.o.), RsrII (5896 n.o.), SfiI (5133 n.o.), SgrAI (2220 n.o.), StuI (5184 n.o.), Tth111I (5496 n.o.), XhoI (3343 n.o.).

For the production of protein was selected cell line Cricetulus griseus Cho DHFR-. The original line acquired in American cell culture collection (ATSS), CRL 9096. The choice of cell line due to the presence of optimal glycosylation profile for the synthesis of human proteins, as well as stability and security of this cell line, which is anashim parameter for the production of therapeutic proteins. On the basis of this cell line was derived cell line Cricetulus griseus SNO 1F8 - producer of recombinant plasminogen activator person, which is derived from the ovary of an adult Chinese hamster.

Cell line Cricetulus griseus SNO 1F8 characterized by the following features.

Morphological features: the Cells have a typical morphological features of epithelial culture and source are adherent culture. The line is hypodiploidy (2n=22), the modal chromosome number 20.

Cultural characteristics: the Cells are cultured in a monolayer on simple nutrient media, for example, on the environment DMEM in the presence of 2-10% fetal serum.

Physiological and biochemical characteristics: the Cells grow at 37°C, pH 6.7-7.3.

Antibiotic resistance: the Cells are resistant to geneticin at a concentration of 1 mg/ml

Strain cell line Cricetulus griseus SNO 1F8 was deposited in the Russian national Collection of industrial microorganisms in WRATH Genetics-PMBC number H-127, the date of Deposit of 20.06. 2012

The claimed invention is illustrated in the following figures:

Figure 1 shows the amino acid sequence of tPA protein, SEQ ID NO:1.

Figure 2. presents the optimized nucleotide sequence of the tPA gene, SEQ ID NO:2.

Figure 3 (a) and (b) presents the scheme for vector design the products pBK415 for the expression of recombinant tPA

Figure 4 presents a map of the recombinant plasmid DNA pBK-415 used to create producer of recombinant tPA.

Figure 5 presents the allocation of recombinant tPA.

The invention is illustrated by the following examples.

Example 1. Construction of recombinant plasmid DNA pBK415.

The nucleotide sequence corresponding to the cDNA of the gene of tissue plasminogen activator person, get chemical-enzymatic synthesis. For this optimized gene sequence is divided into overlapping fragments of a size of about 50 BP Chemical synthesis of oligonucleotides corresponding to these fragments is performed using a solid-phase fosfolipidnogo method, for example, using a synthesizer ASM-102U (BIOSSET Novosibirsk, Russia). The resulting oligonucleotides were subjected to 5'-terminal phosphorylation using T4 polynucleotide kinase (NEB, USA). Phosphorylated oligonucleotides were mixed in equimolar ratio in 50 μl of buffer containing 20 mm Tris-HCl, pH 7,56, 10 mm MgCl2, 0.2 mm rATP, 10 mm dithiothreitol, heated to 65°C, slowly cooled to 37°C for one hour and add T4-DNA ligase. The reaction ligating DNA is carried out for 12 h at 37°C, 0.1 µl of the resulting solution used as matrix in the polymerase chain reaction (NDP) in the presence of thermostable DNA-p is limarzi Pfu and specific primers:

F7_fwd

5'-TAGGCTAGCCGCCACCATGGACGCCATGAAGCGCGGCCT-3' and

F7_rev 5'-CGACGCGTTAGGGGCGCATGTTGTCGCGGATCCA-3'.

Synthesized thus the DNA fragment contains the cDNA sequence of the gene tissue plasminogen activator person, flanked by recognition sites of restricted NheI and MluI - SEQ ID N2 (figure 2). The product of amplification hydrolyzing restrictase NheI and MluI, purified by electrophoresis in 1% agarose gel, the DNA band size of approximately 1700 BP isolated from the gel by the method of electroelution and precipitate DNA from solution by ethanol.

To obtain the vector DNA pBK415 used a series of successive stages of the clone (Figure 3 a) and b)).

As the original plasmid was selected vector pIRES (Clontech, USA). Using the restriction sites XbaI and NotI, in the specified vector cloned gene digidrofolatreduktazy mouse obtained by PCR using genomic DNA isolated from murine myeloma cells SP2/0, and specific primers

DHFR_fwd

5'-CCTCTAGATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAG-3'

and DHFR_rev

5'- GGAAGCGGCCGCTTAGTCTTTCTTCTCGTAGACTTCA-3'

followed by site-directed mutagenesis to optimize the consensus sequence surrounded by initiation codon IRES element with a set QuikChange® II XL (Stratagene, USA) and the following oligonucleotides:

D3_fwd

5' - CCTTTGAAAAACACGATGATAATATGGCCACAACCATGGTTCGACCATTGAAC

TGCATC-3'

and D3_rev

5' - GATGCAGTTCAATGGTCGAACCATGGTTGTGGCCATATTATCATCGTGTTTTT CAAAGG-3'.

In the resulting plasmas which do RAR the restriction sites NheI and MluI cloned previously synthesized cDNA sequence of the gene of tissue plasminogen activator. The selection obtained after transformation of the ligase mixture of bacterial cells XL-10 Gold held in such a way as to detect plasmid DNA pBK413 containing the cDNA sequence of tissue plasminogen activator person.

Plasmid pBK413 then sequentially treated with restriction enzyme Bglil and T4 DNA polymerase, and the resulting vector was used in a ligation reaction with the PCR product obtained with the use of MAR-specific primers

MAR_fwd 5'-GGGGATCCGTAATACAATTGTACCAGGTTTTGGTTTATTAC-3' and

MAR_rev 5' -GAAAACAATATATTTCCAAATGAAAAAAAAATCTGATAAAAAG-3'.

The selection obtained after transformation of the ligase mixture of bacterial cells XL-10 Gold held in such a way as to detect plasmid DNA RVC containing the MAR sequence in the genomic orientation relative to the promoter.

Further, the primary sequence of the obtained plasmid DNA was confirmed by the method of sequencing by Sanger. According to the sequencing of the selected plasmid, the nucleotide and corresponding amino acid sequences NheI-MluI fragment which is fully identical to the cDNA sequence of the gene tissue plasminogen activator person, which was used in further work.

Example 2. Obtaining cell line Cricetulus griseus CHO 1F8, consistently producing recombinant plasminogen activator person.

To acquire the FL cell lines, consistently producing recombinant plasminogen activator person (rtPA), cell line Cricetulus griseus CHO DHFR-(ATCC CRL 9096), transferout plasmid DNA RVK, linearized by BspHI restriction site, for example by the method of lipofectin using reagent jetPEl™ (Polyplus Transfection Inc., USA).

Cells Cricetulus griseus CHO DHFR seeded in wells sectionone tablet (e.g. Costar) with a density of 250×103cells/well in 3 ml of medium DMEM/F12 containing 10% serum, and incubated 24 hours at 37°C in CO2the incubator.

Then hold transfection, using 2 µg of the linearized by BspHI restriction site of plasmid RVC and 4 ál jetPEl™ per well according to the manufacturer's recommendations (the ratio N/P=5). 48 hours after transfection start breeding culture in order to obtain stable transfectants by changing the environment on selective: MEM medium with addition of 10% cialisbuynow serum in the presence of antibiotic geneticin at a concentration of 1 mg/ml Change in the selective environment regularly every 3-4 days. Starting from the 7th day, the wells are examined for the presence of clones of actively dividing cells. Upon completion of the selection process (end of selection was determined by the total loss of not transtitional cell population, which is also under pressure antibiotic) and restore vitality pool of stable transfectants was the definition of who and its productivity.

The productivity of clones evaluated by the method of enzyme-linked immunosorbent assay (ELISA), using, for example, set Max Assay (AssayPro, USA). To do this in the wells in which the cells reached 80-90% confluently, replace the medium with fresh and incubated for 24, 48 and 72 hours at 37°C in CO2-incubator. Supernatant used in ELISA test. The productivity of the pool of stable transfectants was 25 µg/L.

To increase the level of expression of recombinant plasminogen activator human pool of stable transfectants was subjected to the procedure of amplification with methotrexate (MTX).

For biotechnological purposes in the system amplification of recombinant genes as a breeding marker often used gene digidrofolatreduktazy (DHFR), integrated in expressing vector. DHFR catalyzes the conversion of folic acid to tetrahydrofolate, required for the synthesis of eukaryotic cells glycine, timeintensity and purine bases. In this regard, cells SNO, in which the DHFR gene inactivated by the mutation (Cho DHFR-), do not grow in a medium without nucleosides and acquire this ability after transfection DHFR gene.

Growing transfectants further selected on the basis of amplification of DHFR gene on the background of increasing concentrations of methotrexate is an inhibitor of DHFR, in a nutrient medium, as the increase in h is s'la copies of the gene will make the cells resistant to the inhibitor at high concentrations. After conducting multiple rounds of selection get a cell population containing up to several hundred copies of the DHFR gene.

For a given stable transfitsirovannykh pool was held on 4 consecutive rounds of amplification with methotrexate. For this purpose, the cells are cultured in MEM medium containing glycine, gipoksantin and thymidine in the presence of 10% cialisbuynow fetal serum with the addition of increasing amounts of methotrexate (table 1). The duration of each round is approximately 10-14 days.

Table 1
The concentration of MTXProductivity pkg/CL/dayProductivity, mg/l
Without MTX0,0090,025
200 nm0,120,15
400 nm0,141,0
600 nm3,68,0
800 nm15,09,9

Next amplificatory pool stable Tr is stakanov subcloning method of limiting dilution. For sublimirovanny culture rasshivaetsya with a density of 0.5 cells/well in 96-well plates. Just registered and analyzed 130 clones.

Table 2
ClonesProductivity: monoclona culture medium MEM+10% serum
pkg/CL/daymg/l
1B14,515,8
1F83,59,8
1G44,6to 12.0
W3,06,4
3 ÷ 46,819,0
3G92,510,4
2D111,78,8
1D74,48,3

Cell line Cricetulus griseus IF CHO 8 - producer of recombinant plasminogen activator h the rights, was successfully adapted to serum-free suspension conditions of cultivation.

For adaptation procedure was used Protocol gradual reduction of nutrient medium with serum in favor of serum-free medium. The reduction was carried out according to the following scheme:

the ratio of medium with serum and serum-free medium 50/50, then, after stabilization of the cultural vitality of at least 80% reduction of serum environment up to 25%cultivation to stabilize the viability and, further, the transition to the ratio of 10/90 and finally to 100% serum-free medium. Cultivation on a shaker in 100% serum-free medium was performed to stabilize the viability of the culture at the level of 90-95% and doubling time.

Characteristics of this line are presented in Table 3:

Table 3
Cultural environment: SFM4CHO (Hyclone), suspension cultivation
Productivity, mg/l/day14-19
Specific products, pkg/cell/day13,5
The doubling time, h28-30

This clone was after analyzing the stability producing ability and growth characteristics. For this purpose, cells were cultured for 50-100 generations, periodically evaluating the level of expression of rhtPA and growth characteristics of the clones.

Optimization of nutrient medium composition has led to an increase in the level of expression of the target protein to about 50 mg/l/24 hours So that the cultivation of this clone in the presence of specific nutritional supplements SNO Bioreactor supplement (SAFC Biosc.) leads to increase production by 1.5 times, and in combination with sodium butyrate (0.5-1 mm) or dimethysulfoxide (1% V/V) increase in specific products is 2-2,5 times.

Thus, were one of the most productive and stable clones Cricetulus griseus CHO 1F8, which was then used to create a master-Bank and the subsequent production of recombinant plasminogen activator person.

Example 3. Cultivation of cells-producers rhtPA in the bioreactor with the perfusion medium.

Cell suspension cultured in the bioreactor with perfusion of culture medium in a volume of 0.2 to 1 volume of the bioreactor/day, typically 0.25 to 0.5 volume of the bioreactor/day at 37°C, pH 7.0±0.2, a content of dissolved oxygen in the range from 20 to 50% saturation with 5% CO2in the gas phase.

For growing cells-producers in the phase of logarithmic growth using nutrient medium SFM4CHO; productive period is in the stationary growth phase in culture medium additives contribute: SNO Bioreactor supplement (SAFC biose to) 5 ml/1 l of nutrient medium per day, dimethyl sulfoxide 1%V/V glucose to the level of not less than 3 g/L.

When the concentration of cells in the stationary growth phase in the 5-15 million/ml, typically 10 million/ml, the expression of the target protein tPA is 70-190 mg/l of medium per day, usually 150 mg/l/day.

Example 4. The allocation of tPA using metal-chelate chromatography IMAC Sepharose.

IMAC-sepharose, intense ions of the transition metal is pseudoephrine adsorbent for proteins with specific binding of the corresponding metal. The tPA molecule contains a binding site of zinc that allows you to specifically adsorb the protein on the Zn-IMAC-sepharose. Therefore, the selection on the IMAC-sepharose used to capture a fraction of tPA from the culture fluid and clean it from extraneous proteins that do not possess affinity for transition metals and does not bind to the sorbent. This method also allows you to concentrate protein before applying for the following chromatographic stage.

All chromatographic stage of selection was carried out at a temperature of 4-10°C, buffer solutions pre-cooled. Before applying cultural liquid sorbent was saturated zinc ions by passing through the layer of sorbent 0.2 M solution of zinc acetate, followed by washing with water and balanced starting buffer (20 mm Tris-HCl, 100 mm NaCl, 0.05% Polysorbate 80, 5% alcohol (isoprop levy or ethyl), pH 8.0).

After the application of cultural liquid sorbent was washed from unbound impurities of the starting buffer, then washed with high salt buffer (containing 1M NaCl), imidazole buffer (containing 0.1 m imidazol) and nitrosonium buffer (containing 50 mm sodium acetate). These leaching allow to separate tPA from nonspecific and weakly bound impurity proteins and nucleic acids of the producer strain, and also to prepare a product for elution.

The fraction of tPA was suirable acidic buffer containing 50 mm EDTA and diluted 5-fold sour nitrosonium buffer containing 50 mm sodium acetate (pH 4.5). The resulting solution was used for application to the next stage of the selection.

Example 5. The allocation of tPA using katiana-exchange chromatography on SP Sepharose with precolonial Q Sepharose

When selecting tPA at this stage used serially connected column of Q Sepharose and SP-Sepharose (the ratio of the volume of sorbents about 1:10). Q and SP sepharose are, respectively, strong anyone and katiana-exchange sorbents, allowing the separation of biomolecules with different surface charges; Q sepharose has a high DNA-binding properties. Thus, the simultaneous application of these sorbents at low pH values in the process of giving tPA allows you to effectively remove about is enough DNA producer strain and proteins, possessing a negative charge at the working pH, without loss of the target protein (Q sepharose) and quantitatively adsorb tPA (SP-sepharose)optionally purifying the protein from impurities and turning it into a suitable buffer solution for subsequent chromatographic stage.

Columns with sorbent pre-balanced starting buffer (50 mm sodium acetate, 100 mm NaCl, 0.05% Polysorbate 80, pH 5.0), and then inflicted dilute the eluate with the IMAC sepharose. The fraction of tPA was suirable alkaline buffer containing 1M isothiocyanate potassium, and diluted 10 times nitrosonium alkaline buffer (containing 100 mm Na-CO3, pH 10.0). The resulting solution was used for application to the next stage of the selection.

Example 6. The allocation of tPA using pseudoephrine chromatography on Lysine Lysine Sepharose of separase is pseudoephrine adsorbent for proteins having a binding region (biospecific or local charge) with lysine. Monomeric molecule specific tPA adsorbed on the Lys sepharose, while the oligomeric form is not contacted by the media that can effectively separate oligomeric forms from the monomer and to obtain a concentrated fraction of protein in the lysine-containing buffer solution, stabilizing the product in Monomeric form.

A column of Lysine by separate pre-balanced starting buffer (20 m is Na-CO 2, 0.1 M NaCl, 0.05% Polysorbate 80, pH 10.0), and then put the diluted eluate obtained in the previous example. The fraction of tPA was suirable buffer containing 50 mm lysine.

Example 7. The concentration and diafiltration tPA in the buffer ready form.

For diafiltration used polyethersulfone filter impermeable to particles weighing more than 30 kDa, which was previously balanced buffer prepared of the following composition: 400 mm arginine-phosphate, 0.02% Polysorbate 80, pH 7.3. In the eluate obtained in Example 6 was made of dry L-arginine hydrochloride to a concentration of 400 mm. After complete dissolution, the resulting solution was concentrated on a system for diafiltration to the concentration of 4-5 mg tPA/ml, then diafiltrate in the buffer ready form by passing through a system of 8-10 volumes. The obtained substance was frozen in liquid nitrogen and kept at -80°C.

Example 8. Measurement of the biological activity of the substance tPA.

The determination of the activity of tPA was performed by three methods - using a direct chromogenic substrate tPA; using chromogenic plasmin substrate (indirect substrate and method of lysis of fibrin clots (LFS). In each of the methods as the standard used international standard activity of tPA and was the calibration scale for determining the activity.

In the first method to tPA was added to the nternet synthetic peptide substrate, coupled with the chromophore, resulting in cleavage of the substrate. As a result of this reaction mixture had become yellow. Measured absorption of the reaction mixture at 405 nm, which is directly proportional to the activity of tPA.

When using the second method, the reaction mixture was added to the tPA, the natural substrate of tPA, plasminogen, and plasmin substrate (product of cleavage of plasminogen), coupled with the chromophore. In the result of the activation of plasminogen resulting plasmin were digested substrate, and the released chromophore resulted in staining solution in yellow color, the intensity of which is directly proportional to the activity of tPA.

When using the third method in the reaction mixture were added the components necessary for the formation of fibrin clot (normal plasma and kaolin), and the components necessary for lysis (plasminogen and tPA), and measured the time of lysis, which is proportional to the activity of tPA. In this case, the measurement of the activity of tPA is based on the ability of tPA to activate plasminogen to plasmin to fibrin clot that leads to its lysis.

The specific activity of the substance tPA determined using the above methods, was 580000 IU/mg.

Thus, the present invention allows to obtain a recombinant plasminogen activator person with identical properties St is Istvan natural polypeptide. Received rtPA can be used for the manufacture of medicinal preparations and pharmaceutical industries.

1. Recombinant plasmid DNA RVC encoding a polypeptide with the sequence of tissue plasminogen activator human sized 11383 BP, physical map presented on figure 4, and consisting of the following elements:
- optimized sequence encoding the polypeptide of tissue plasminogen activator person, SEQ ID NO: 2,
- MAR is the area of attachment to the nuclear matrix gene lysozyme birds,
amplifier transcription of the virus CMV,
- internal site of translation initiation IRES virus encephalomyocarditis,
gene DHFR mouse
- polyadenylation signal of the SV40 virus,
- aminoglycoside-3'-phosphotransferase, providing resistance strain cells-producer to geneticin (Neo),
cassettes for expression in bacterial cells the gene of β-lactamase, providing resistance to ampicillin,
unique recognition sites for the following restriction endonucleases: AhdI (7352 BP), AsiSI (508 BP), BglII (2649 BP), BipI (2079 BP), BmtI (654 BP), BssHII (5778 BP), BstBI (6062 BP), BstEII (2388 BP), Bsu36I (2245 BP), EcoRI (10035 BP), EcoRV (10969 BP), FspAI (2020 BP), luI (2433 BP), MreI (2220 BP), NheI (654 BP), NotI (3918 BP), PspXI (3342 BP), RsrII (5896 P.O.), SfiI (5133 P.O.), SgrAI (2220 po), StuI (5184 BP), Tth111I (5496 BP), XhoI (3343 BP).

2. Cell line Cricetulus griseus CHO 1F the producer of recombinant tissue plasminogen activator person, obtained by transformation of the cell line Cricetulus griseus CHO DHFR - recombinant plasmid DNA RVC according to claim 1.

3. The method of acquisition and allocation of polypeptide having the activity of tissue plasminogen activator, comprising culturing the cell line of claim 2, where the expression of the specified polypeptide cultivation is carried out in perfusion conditions in the presence of a mixture of additives CHO Bioreactor supplement and sodium butyrate or dimethyl sulfoxide, and the secretion of the polypeptide having the activity of tissue plasminogen activator.

4. The method according to claim 3, where in the composition of the mixture used is 0.5-1.5 mm sodium butyrate or 1% (V/V) dimethylsulfoxide.

5. The method according to any of PP or 4, where the cell line adapted to a serum-free environment.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes compounds of labyrinthpeptins A1, A2, or A3 of formula (I) , where {A}, {B}, {C}, R1-R6, m and n have the values specified in the formula of the invention. Compounds are obtained at fermentation of Actinomadura namibiensis DSM 6313 strain under acceptable conditions in cultural environment till one or more compounds of formula (I) are formed. The invention proposes the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A2, and the deoxyribonucleic acid (DNA) coding preprolabyrinthpeptin A1, as well as preprolabyrinthpeptins A1 and A2, and prolabyrinthpeptins A1 and A2. Labyrinthpeptins of formula (I) are used for therapy of infections caused by gram-positive bacteria, virus infections and/or neuropathic pain caused by inflammation.

EFFECT: improving therapy efficiency.

24 cl, 4 tbl, 20 ex

FIELD: biotechnology.

SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.

EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.

16 cl, 2 dwg, 4 tbl

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

FIELD: biotechnologies.

SUBSTANCE: plasmid genetic structure pOL-DsRed2 is produced, being built on the basis of a plasmid vector pIRES (Clontech), where fragments of cDNA of human genes OCT4 and LIN28 are placed, being connected with a nucleotide sequence coding P2A-peptide and gene cDNA, coding fluorescent protein DsRed2.

EFFECT: invention provides for simultaneous translation of human proteins OCT4 and LIN28 and fluorescent protein DsRed2 in production of induced pluripotent stem cells of a human being and animals in medicine and veterinary science.

3 cl, 1 dwg, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. Disclosed is a method of isolating and purifying recombinant human growth hormone which is secreted by Saccharomyces cerevisiae yeast during fermentation thereof in suitable conditions. The target protein is precipitated in biomass-free culture fluid by either acidification to pH 2.9-4.0 or adding polyethylene glycol with molecular weight of 3000-6000 Da. The obtained precipitate is then dissolved in a suitable solvent. Preliminary purification of the target protein is carried out either by anion-exchange chromatography at pH 5.6 or by diafiltration in the presence of 0.1-0.5 M sodium chloride. Main purification of the target protein is then carried out by anion-exchange chromatography at pH not below 7.3 and gel filtration.

EFFECT: invention enables to obtain a growth hormone which is free from parent proteins, host-producer protein and other impurities such as pigments, with output of up to 60%.

8 ex

FIELD: biotechnology.

SUBSTANCE: method provides for culturing mammalian cells expressing the recombinant protein of interest in a culture medium containing anti-aging compound selected from carnosine, acetyl-carnosine, homo-carnosine, anserine, and K-alanine and their combinations, under conditions and for a time sufficient for expression of the protein.

EFFECT: invention enables to improve the performance of the cell culture selected from higher titer, of increased specific cell productivity, increased cell viability, increased integrated viable cell density, decreased accumulation of high molecular aggregates, decreased accumulation of acidic molecules, and their combinations.

44 cl, 14 dwg, 3 ex

FIELD: biotechnologies.

SUBSTANCE: method consists in expression of a gene of a human plasminogen fragment from 453 to 543 amino acid within E.coli cells with subsequent extraction and treatment of a finished product from cell periplasm. Expression is carried out in cells E.coli BL21 (DE3), transformed by plasmid DNA pEK5 or pEK5H with physial maps represented in figure 2, containing a gene of target polypeptide fused with a gene of signal peptide OmpA, origin of replication pUC ori, a gene of resistance to kanamycin and a gene coding lac-repressor under control of T7-promotor. At the same time the recombinant plasmid DNA pEK5H additionally contains between genes OmpA and target polypeptide the sections coding amino acid sequences HHHHHH and DDDDK.

EFFECT: invention provides for secretion of target polypeptide into cell periplasm with high yield.

4 cl, 5 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

FIELD: biotechnologies.

SUBSTANCE: invention proposes an antibody that specifically connects segment M1' IgE and that induces apoptosis in IgE-expressing B-cells and its antigen-binding fragment. Besides, compositions and curing methods of IgE-mediated abnormalitiy, an item, a specific elimination method of IgE-producing B-cells, methods for prophylaxis and reduction of IgE products induced with an allergen, as well as isolated nucleic acid, an expression vector, a host cell and a method for obtaining an antibody as per the invention together with their use are considered.

EFFECT: invention can be further used in therapy of diseases associated with IgE.

46 cl, 19 dwg, 5 tbl, 13 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: proposed chimeric protein with SEQ ID NO:02 is fluorescent biosensor, built on the basis of HyPer protein and mutant of PH-domain of Btk tyrosine kinase.

EFFECT: proposed inventions allow performing simultaneous monitoring of product of hydrogen peroxide and phosphatidyl inositol-3,4,5-triphosphate in a living cell.

4 cl, 4 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: present inventions relate to protein engineering, plant molecular biology and pest control, as well as a hybrid insecticide protein and use thereof. Described is a hybrid insecticide protein which includes from the N-end to the C-end an N-end portion of Cry3A protein which is fused with the C-end portion of Cry1Ab protein, wherein the position of the crossover of the Cry3A protein and the Cry1Ab protein is located in a conservative block 2, in a conservative block 3 or in a conservative block 4 and has anti-western corn rootworm activity. Also disclosed are nucleic acid molecules which code the novel proteins, methods of producing proteins, methods for use thereof, as well as transgenic plants and seeds thereof which contain such proteins.

EFFECT: inventions enable to obtain cheap means of controlling Diabrotica worms.

39 cl, 8 dwg, 9 tbl, 46 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to immunology. What is presented is a humanized human monoclonal CD19 antibody prepared of an HB12B antibody, or a fragment thereof characterised by amino acid sequences of variable domains. Also, there are presented nucleic acids coding polypeptides having the sequences of the variable domains, and a cell expressing the antibody under the invention, and a pharmaceutical composition, and a method for treating a B-cell diseases or disorders in a human.

EFFECT: invention can find further application in treating various CD19-associated diseases, including autoimmune diseases, and preventing or treating the graft-versus-host disease (GVHD), and the humoral rejection and post-transplantation lymphoproliferative disorder in a human graft recipient.

21 cl, 45 dwg, 40 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant nucleic acid expresses one or several polypeptides of interest, a vector of expression and bacteria, which contain this recombinant nucleic acid. The recombinant nucleic acid contains a natural promotor of a gene of HU-like DNA-binding protein (PhilA) of Lactococcus type with the sequence SEQ TD NO:28, or its homological or functional version, which at least by 95% identical to the promotor with sequence SEQ ID NO:28, functionally linked with one or several open reading frames, heterological for the promotor RhIIA, where the promotor RhIIA is located above one or several open reading frames. The expression vector contains the above recombinant nucleic acid, preferably, the specified vector is produced from pTINX. A bacterium contains the above recombinant nucleic acid or the above vector.

EFFECT: proposed invention makes it possible to increase level of expression of polypeptide genes of interest and therefore produce sufficient number of expressed proteins.

19 cl, 26 dwg, 12 tbl, 9 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biochemistry and immunology. What is considered is an isolated antibody molecule which binds an alpha chain of human granulocyte/macrophage colony stimulating factor receptor (GM-CSFRα) and inhibits GM-CSF binding to human GM-CSFRα. There are presented: a composition for GM-CSFRα inhibition or neutralisation and a composition for treating rheumatoid arthritis, asthma, chronic obstructive pulmonary disease or myeloid leukaemia, containing the antibody under the invention; the isolated nucleic acid molecule coding the antibody under the invention, a host cell and a method for preparing the antibody under the invention, as well a method for inhibiting or neutralising GM-CSFRα activity and a method of treating rheumatoid arthritis, asthma, chronic obstructive pulmonary disease or myeloid leukaemia in a patient.

EFFECT: invention can find further application in therapy of the GM-CSFRα-mediated diseases.

41 cl, 5 tbl, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers biotechnology and medicine. What is described is an immunogen for inducing the IgE immune response. The immunogen contains at least one IgE antigen peptide bound to an immunogenic carrier. The immunogenic carriers may be presented by virus-like particles specified in a group consisting of HBcAg, HBsAg and Qbeta VLP There are also disclosed compositions and method for preventing, relieving or treating an IgE-associated disorder in an individual with using the above immunogen.

EFFECT: group of inventions may be used in medicine for treating the allergic diseases.

20 cl, 2 dwg, 13 tbl, 14 ex

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant blood coagulability factor IX of human being (hFIX). Recombinant plasmid DNA pAK380 containing gene of protein rhFIX, MAR - binding area to nuclear matrix of lysozyme gene of birds, virus transcription enhancer CMV and an internal translation initiation site IRES of encephalomyocarditis virus, gene DHFR of a mouse, a polyadenylation signal of virus SV40, gene of aminoglycoside-3'-phosphotransferase for stability to geneticin (Neo), a cassette for expression in bacteria cells of gene β-lactamase for stability to ampicillin, is used for obtaining recombinant factor hFIX in cells of line Cricetulus griseus CHO 1E6. By transformation of cell line C. griseus CHO DHFR - recombinant plasmid DNA pAK380 there obtained is cell line C. griseus CHO 1E6 producing recombinant hFIX with stable high yield at the level of 50 mg/l/24 h. After cultivation of cells-producers there extracted is hFIX by pseudoaffine chromatography on Q Sepharose with elution of 10mM CaCl2; then, on Heparin-Sepharose FF with elution of 600 mM NaCl, and chromatography on hydroxyapatite of type I with elution of 600 mM K3PO3 and chromatography on Source 30Q with elution of 600 mM with ammonium acetate.

EFFECT: improvement of the method.

4 cl, 5 dwg, 7 ex, 3 tbl

Up!