Recombinant plasmid dna pap227 coding polypeptide of recombinant factor viii of human blood coagulability, line of cells cricetulus griseus cho, 2h5-producer of recombinant factor viii of human blood coagulability, and method for obtaining polypeptide having activity of recombinant factor viii

FIELD: biotechnologies.

SUBSTANCE: invention can be used for obtaining recombinant human blood coagulability factor VIII with deletion of B-domain (hFVIII-BDD). Recombinant plasmid DNA pAP227 coding polypeptide with sequence hFVIII-BDD 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 2H5 producing recombinant hFVIII-BDD with highly stable yield at the level of about 20 IU/ml/24 h. Cultivation of cells-producers is performed in medium DME/F12 containing 2-4% of Fetal Bovine Serum, 1% of dimethylsulphoxide and 50 IU/l of insulin.

EFFECT: improvement of the method.

4 cl, 5 dwg, 9 ex

 

The technical field

The invention relates to biotechnology, in particular genetic engineering, and can be used to produce recombinant factor VIII clotting person (FVIII). The invention is a recombinant plasmid DNA RAR encoding the polypeptide sequence of recombinant factor VIII clotting person with a deletion In the domain, and producer based cell line Cricetulus griseus CHO 2H5 containing the indicated plasmid DNA synthesizing recombinant factor VIII clotting person with a deletion In the domain.

Prior art

Deficiency of factor VIII leads to the development of hemophilia a, the most common form of this disease, which constitutes about 85% of all cases of hemophilia. The incidence of hemophilia a, according to the who, ranges from 5 to 10 cases per 100 thousand newborn boys. In Russia, according to the National hemophilia Foundation, the disease affects about 10,000 people. The most characteristic symptom of hemophilia is bleeding, occurring even after a minor injury. Often hematoma, marked joint hemorrhage, possible bleeding in the bone tissue, leading to necrosis and the reason for bone decalcification.

For normal hemostasis level of factor VIII should be not less than 25%, but Matica usually appears when it is reduced to 5% and below. The severity of the disease is directly related to the level of the factor.

The essence of the treatment of hemophilia a is reduced to replacement therapy with factor VIII, therefore, the establishment of appropriate and effective ways to get that protein is one of the most important tasks of biotechnology.

Currently, the factor VIII is an engineered method using mammalian cells, precluding the need to use the factor of blood obtained from the plasma of donors and the risk of the relevant viral infections is minimized.

Factor VIII - high molecular weight glycoprotein present in blood in the form of inactive cofactor. He contributes to the activation of factor X protease produced by the internal mechanism of coagulation. Factor VIII is synthesized by hepatocytes and circulates in plasma in a complex with factor a background of Villebranda.

Factor VIII is a large-size protein that includes six domains: A1(1-336)-A2(372-710)-(741-1648)-A3(1896-2019)-C1(2020-2172)-C2(2173-2332) and consists of 2332 amino acids after cleavage of the signal peptide), with a complex profile of glycosylation. Industrial production and stabilization of such a large protein very difficult and expensive. It is not surprising, therefore, that a significant part of research in recent years has been aimed at identifying modified forms of factor VIII, to whom that with decreasing size of the protein would preserve the level of procoagulant activity, inherent natural factor VIII clotting of blood.

The result of these studies it was found that the form factor VIII, with divisions in the field of In-domain exhibits a coagulation activity such as protein source (for example, see Biochemistry, 1986 Dec 30; 25(26):8343-7; Biochem J. 1991 Jul 1, 277 (Pt1):23-31; Eur J Biochem. 1995 Aug 15, 232(1): 19-27; Semin Hematol. 2001 Apr, 38(2 Suppl 4):4-12).

In addition, removal of In-domain reduces the number of antigenic determinants by the disappearance of part of the sites of N-glycosylation, which in turn reduces the risk of appearance of specific antibodies in a patient receiving therapy with factor VIII.

The modified factor VIII with a deletion In the domain (rhFVIII-BDD) is a protein with a mass of about 170 kDa, consisting of two fragments, the size of 90 kDa and 80 kDa, respectively. While maintaining the same level of biological activity, it is more stable than the full-size factor VIII. In particular, the drug Refacto made on the basis of the truncated protein does not require the presence of albumin to stabilize. Furthermore, the reduction in size of the protein can increase the level of expression of about 10 times compared with the full-length protein (patent US 5661008).

However, it is noted (for example, see patent US 6358703)that even for factor VIII with delegated domain, expression in mammalian cells usually 2-3 of order n is the same than the expression of other proteins using similar recombinant vectors and techniques. For these reasons, the task of creating cell lines, allowing to achieve a high level of expression of factor VIII clotting of blood, remains.

To achieve high levels of expression are used in different ways, from different variants of optimization of the FVIII protein sequence and structures of expression vectors to the selection of the most effective cell lines. The most preferred are mammalian cell lines, to achieve correct from the point of view of preserving the functions of the recombinant protein set it posttranslational modifications, as well as elements of its secondary and tertiary structures. Size deletional sequence in the field of In-domain varied works in the range of from about 500 to 1000 amino acids. In addition, to optimize the sequence used point mutations of amino acids and different length linkers (from 3 to 25 amino acid residues) between circuits 90 and 80 kDa.

For example, 'toole with employees, reports about expresii FVIII, not containing portions of the sequence from 982 to 1562 and from 760 to 1639 amino acids, respectively (Proc. Nati. Acad. Sci. USA, Vol.83, pp.5939-5942, August 1986, Biochemistry). Such FVIII was 10-20 times more Polarstern the th FVIII wild-type.

In the already mentioned work (Biochemistry, 1986 Dec 30; 25(26):8343-47) were created vector containing several types of FVIII sequences: the first type included the site In the domain, since Arg 747, Arg752, or Arg776 attached to Glu 1649; the second type did not contain the area between Ser743 and Gin1638 and included the linker of the 14 amino acids; in the third type of structures In the domain has been removed completely or replaced by the linker from 1-4 Arg residues. Significant expression level (0.5-1 IU/ml) was achieved only in the case when the domain was replaced by 3 or 4 Arg residue.

Design with divisions in the field In the domain is also described in EP 295597. Was removed a section of the domain between 816 and 1598, and between 741 and 1689 amino acids. The authors noted that the half-life of such mutant forms comparable to the full-length protein, and activity against thrombin even higher than that. The level of expression clones in cells SNO and KSS was about 8-15 ME/ million cells/ ml

Patent US 5112950 offers constructs containing deletions in the field 868-1562 and 771-1666. The last option (with a deletion in the field 771-1666) in the test thromboplastin was 5 times more, and the level of its production was 10 times higher compared to the baseline FVIII.

Patent US 4868112 reveals the construction of vectors containing FVIII gene without a specific remote area in the field of In-domain (ranging in size from 581 to 915 amino acids) and the gene for dihydrofolate reductase (DHFR), and DHFR-deficient cell line of Cho, transfetsirovannyh these vectors. The expression level of the protein in this system was about ME/ml/day.

Patent US 5661008 describes several variants of factor VIII with delegated domain (sites 741-1648 and 745-1562), and the remaining part of the protein in several variants were connected arginine by linkers of different lengths: from 2 to 4 aminokislotami believe that the linker is longer than 10 amino acid residues are undesirable. The expression of the received vector constructions were carried out in Cho cells, the expression level was 0.8-1.5 IU/ml (for different versions).

In the patent RU 2429294 described system the expression of factor VIII with a deletion In the domain in cells SNO, not containing proteins of animal origin intended for use in biopharmaceutical production. The productivity of this system is 0.5 Units/million cells/day. However, the stability was confirmed in only three passages, it is insufficient for the industrial production of protein.

In the patent EA considered getting variants of factor VIII with a deletion In the domain, replaced by Arg-rich linker peptide, a length of 10 to 25 amino acid residues, as well as mutants on the basis of such a shortened protein. Expression is performed in immortalized human cells 293 T cells embryonic human kidney), the ri together with the target protein is the expression of at least one viral protein activator transcriptio. Because the medicine on the basis of Factor VIII suggests a systematic introduction into the bloodstream of the patient, and the admixture of viral transcripting activator in the preparation unacceptable from the point of view of the significant risk of tumorogenic properties, the production technology of the relevant medicinal product is much more complicated by introducing additional steps in the procedure for the selection and validation of viral purity of the final product. This leads to excessive costs in industrial production.

Patent RU 2249041 describes a method of obtaining cell lines NCV, transfected with the vector pCIS25DTR containing region transcription FVIII-BDD, DHFR as a selective marker, and the end sequence repeat of Epstein-Barr to improve integration. The authors argue that in this way it is possible to obtain a cell line with productivity up to 20 MCAD/KL/day. However, there is no information about the stability of these clones and suitability for industrial use. In addition, the disadvantage of this technical solution is the use of cancer cells (Burkitt lymphoma)that may be unsafe because of the additional risks when working with such cells, a more complicated procedure for isolation and purification of protein, necessary in order to avoid transforming the aktivnosti or impurities tumorogenic material in the final product (US patent 7470523, Int J Cancer 1999 Nov 12; 83(4):555-63).

The main drawback of most cellular systems obtain protein, known from the prior art, is a low level of expression required for the industrial production of factor VIII. In addition, the presence of mutations and artificial unnatural sequences (linkers) in the protein can result in a risk of occurrence of an immune response against the drug on the basis of this protein, which is highly undesirable.

In an effort to minimize these risks, we have selected for expression of the sequence factor VIII, in which the linker insert is missing completely, and the plot deletirovanie In-domain represents amino acids 743 in 1638 (see figure 1). It is this sequence of rhFVIII-BDD was used only in the present preparation with a delegated domain, Refacto, which is recognized as effective and safe.

This sequence is described in European patent EP 0506757 and the article "Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization" (Eur J Biochem. 1995 Aug 15, 232(1): 19-27). The corresponding expression system is the closest analogue of the present invention. Protein rFVIII-SQ is a FVIII with a deletion of part of the In-domain and peptide bond between Ser743 and Arg1638. Plasmid pKGE327 containing the DHFR gene, expressives in cells CHO-DG44 in risotti 10% fetal serum. After amplification methotrexate managed to reach the level of expression of 0.5-1.5 IU/ml the Disadvantage of this system expression, like that of the other, described above, is the low productivity. In addition, here, as in the other cited sources do not contain information as to the stability of the resulting system, which appear to be extremely important to assess the feasibility of using such systems in industrial production.

Disclosure of inventions

The present invention solves the problem of constructing plasmid DNA containing a sequence of recombinant factor VIII-BDD, and create a cell line (producer strain), allowing to obtain recombinant factor VIII-BDD in good yield (at a constant level of about 20 IU/ml/day) and maintain its performance for a large number of passages (not less than 60), which is an important and necessary for the industrial production of protein.

The problem is solved by constructing recombinant plasmid DNA RAR consisting of 13973 softwares cell line Cricetulus griseus CHO 2H5, allowing to obtain factor VIII blood clotting with consistently high output, sufficient for the industrial manufacture of a medicinal product based on it.

The introduction of various genetic elements in eukaryotic genome in the composition of recombinant what onstructi, 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, such as 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 rhFVIII-BDD, allows to maintain the level of expression of this protein with the highest stability.

The advantages of the invention consist also in the use of chemical-enzymatic method for the synthesis of the gene rhFVIII-BDD, 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, critical sites, splicing, 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 factor VIII in a nutrient medium, and, accordingly, the final yield of the target product.

Recombinant plasmid DNA RAR contains the following sequence: MAR - district attachment to the nuclear matrix gene lysozyme birds, enhancer of transcription of the virus CMV, cDNA gene rhFVIII-BDD, internal site of translation initiation IRES, a DHFR gene, and the polyadenylation signal of the SV40 virus, cassette that contains all the elements necessary for gene expression of aminoglycoside-3'-phosphotransferase (ARN), providing resistance shtam the cells and producer to geneticin, and cassettes for expression in bacterial cells the gene of β-lactamase, providing resistance to ampicillin. Plasmid RAR (figure 3)encoding the factor VIII polypeptide, characterized by the following features:

- consists of 13973 BP,

- has a molecular mass of 9.1 MDA,

- encodes the polypeptide of factor VIII clotting of human blood, with a deletion In the domain name,

- 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 (10094 BP), AsiSI (660 BP), BglII (2674 n.o.), BmtI (1085 n.o.), BssHII (8520 BP), BstBI (8804 BP), Eco47III (13117 BP), EcoRI (12777 BP), EcoRV (13711 BP), MluI (5474 BP), NheI (1085 BP), NotI (6660 BP), PpuMI (5458 BP), RsrII (8638 BP), SbfI (5105 BP), SpeI (152 BP), Tth111I (8238 BP), XcmI (2096 BP).

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 an important parameter for the production of therapeutic proteins. On the basis of this cell line was derived cell line Cricetulus griseus CHO N - producer re ominotago factor VIII clotting of human blood, which is derived from the ovary of an adult Chinese hamster Cricetulus griseus CHO and characterized by the following features:

Morphological features: the Cells have a typical morphological features of epithelial culture, and the source is adherent culture. This line is hypodiploidy (2n=2), 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 700 μg/ml.

Strain cell line Cricetulus griseus CHO 2H5, was deposited in the Russian national Collection of industrial microorganisms, Institute of Genetics-PMBC number H-126, date of Deposit 20.06.2012,

The claimed invention is illustrated in the following figures:

Figure 1 shows the amino acid sequence of the factor VIII gene with a deletion In the domain, 1457 amino acid residues, SEQ ID NO:1.

Figure 2. presents the optimized gene sequence of human factor VIII with a deletion In the domain, 4371 BP, SEQ ID NO:2.

Figure 3 presents a map of the recombinant plasmid DNA RAR used to create strain-producer of recombina is these factor VIII blood clotting person with a deletion In the domain.

Figure 4 (a and b) presents the scheme for vector design RAR for the expression of recombinant factor VIII blood clotting person with a deletion In the domain.

Figure 5 presents the allocation of the obtained protein.

The invention is illustrated by the following examples.

Example 1.

Construction of recombinant plasmid DNA RAR.

The nucleotide sequence corresponding to the cDNA of the gene coagulation factor VIII human with a deletion In the domain (rhFVIII-BDD) receive 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 using a synthesizer, for example, 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 keresni chain reaction (PCR) in the presence of thermostable DNA polymerase Pfu and specific primers:

F8_fwd

5'-TAGGCTAGCCGCCACCATGATGCAGATCGAGCTGAGCAC -3' and

F8_rev 5'-CGACGCGTTATCAGTACAGGTCCTGGGCCTCGCA-3'.

Synthesized thus the DNA fragment contains the cDNA sequence of the gene rhFVIII-BDD, flanked by recognition sites of restricted NheI and MluI - SEQ ID NO:2 (figure 2). The product of amplification hydrolyzing restrictase NheI and MluI, purified by electrophoresis in 1% agarose gel, the DNA band size of approximately 4380 P.O. isolated from the gel by the method of electroelution and precipitate DNA from solution by ethanol.

To obtain the vector DNA RAR used a series of successive stages of the clone (figure 4). 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'-CCTTTGAAAAACACGATGATAATATGGCCACAACCATGGTTCGACCATTGAACTGCATC-3' and

D3_rev

5'-GATGCAGTTCAATGGTCGAACCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGG-3'.

In the resulting plasmid RAR on sigam enzyme NheI and MluI cloned Sintesi is consistent previously the cDNA sequence of the gene rhFVIII-BDD.

The primary structure of the intermediate vector design RAR in the area of initiating codon IRES element was confirmed by the method of sequencing by Sanger.

Plasmid RAR then sequentially treated with restriction enzyme BgII 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 RAR containing the MAR sequence in the genomic orientation relative to the promoter.

Next, selection of recombinant plasmids was performed in such a way as to identify the vector DNA RAR containing the gene sequence of factor VIII in direct relation to the promoter orientation. 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 rhFVIII-BDD, which was used in further work.

Example 2. Getting lean and cells Cricetulus griseus CHO 2H5, consistently producing recombinant coagulation factor VIII human.

To obtain cell lines stably producing recombinant factor VIII clotting of human blood (rhFVIII), cell line Cricetulus griseus CHO DHFR- (ATCC, CRL-9096), transferout plasmid DNA RAR, linearized by restriction site AhdI, for example by the method of lipofectin using reagent jetPEl™ (Polyplus Transfection Inc., USA).

Cells Cricetulus griseus CHO DHFR - plated on a Petri dish (for example, 10 cm in diameter) with a density of 2×104cells/cm2in 12 ml of DMEM medium containing 10% bovine fetal serum and/or in the holes sectionone tablet (e.g. Costar) with a density of 250×103cells/well in 3 ml of the above medium and incubated 24 hours at 37°C in CO2the incubator.

Then hold transfection using 15 μg of the linearized by restriction site AhdI plasmids RAR and 30 ál jetPEl™ according to the manufacturer's recommendations. 48 hours after transfection start selection on antibiotic geneticin at a concentration of 700 μg/ml Change in selective medium containing the antibiotic, regularly every 3-4 days. Starting from the 7th day, the wells and/or Petri dishes analyze for the presence of clones of actively dividing cells that are resistant to geneticin. Upon completion of the selection process (end of selection was determined by the total loss of not transtitional is opulatio cells, also under the pressure of the antibiotic) Petri dishes and/or sectionone tablets containing stably transtitional culture, were filled with semi-liquid environment Matrix Clone CHO (Genetix) and further cleaopatra occurred in semisolid medium in the presence of antibodies specific for factor 8, labeled with the fluorescent dye Alexa 488. Further subclavian was performed using a robotic system ClonePix (Genetix). They received a total of 150 clones.

The productivity of clones evaluated by analysis using a chromogenic substrate and/or immunoblotting using monoclonal or polyclonal antibodies to coagulation factor VIII human. For this purpose, the holes and/or Petri dishes, in which the cells reached 80-90% confluently, replace the medium with fresh and incubated for 24-72 hours at 37°C in CO2-incubator. The supernatant is used in the test to determine the specific activity using, for example, a set COATEST SP4 FVIII (Chromogenix, Italy) and/or Technoclone, Austria). Also for the detection of FVIII were used a commercial antibody against the heavy chain of factor VIII produced by the company Abeam. The panel leader of the 40 clones was selected for the procedure of amplification with methotrexate.

For biotechnological purposes in the system amplification of recombinant genes as a breeding marker often used in isout 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, so as to increase the number of 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 this leader panel was held on 7 consecutive rounds of amplification with methotrexate. For this purpose, cells of each clone were cultured in IMDM medium containing glycine, gipoksantin and thymidine in the presence of 10% cialisbuynow fetal serum with the addition of increasing amounts of methotrexate: 20 nm, 50 nm, 100 nm, 200 nm, 400 nm, 800 nm, 1600 nm. The duration of each round is approximately 10-14 days. Achieved an increase in productivity of 2.5 to 14 times depending on the clone. The result was the presentation of the s 6 leader clones-producers of rhFVIII, which were subjected to amplification of the higher concentrations of methotrexate (3, 6, and 15 μm), achieved an additional increase in productivity from 6 to 10 times. This leader the next panel was used in the test for stability of the producing ability and growth characteristics. For this purpose, the clones were cultured for 50-100 generations, periodically evaluating the level of expression of rhFVIII and growth characteristics of the clones.

Thus, were one of the most productive and stable clones Cricetulus griseus CHO 2H5, which was then used to create a master-Bank and the subsequent production of recombinant factor VIII clotting of human blood. The level of expression of a protein for a given clone is 9-17 IU/ml/24 h on the environment DMEM in the presence of 10% fetal serum, specific products is 60-80 km mkme/cells/24 hours.

Cell line Cricetulus griseus CHO 2H5 - producer of recombinant factor VIII blood clotting man, was successfully adapted to serum-free suspension conditions of cultivation.

Further cultivation was carried out under different conditions for optimum yield of protein, are described in detail in examples 3 and 4.

Example 3.

Cultivation of cells producing recombinant factor VIII blood clotting person in the roller b is the tyls.

Cells seeded in roller bottles with a density of 5·104CL/cm2in 500 ml of culture medium in the bottle of capacity of 5 l and a surface for the growth of culture cells=2000 cm2. For cell growth using environment DME/F12 with 10% fetal serum.

The cultivation is carried out at 37°C, the content of CO2in the air phase=5% V/V, the speed of rotation of the roller bottle=about 10-20/hour, usually about 12-14/hour.

After the formation of a dense monolayer cultures of cells, typically 72 hours after seeding the cells, replace the medium growth on the environment the product of the following composition: DME/F12 with 2% fetal serum, 400-700 ml of culture medium for 1 roller bottle, typically 500 ml 1 roller bottle.

Daily exercise complete replacement of the volume of the nutrient medium products.

The level of expression of the target protein in these culture conditions is 9-12 IU/ml (or 0.9-1.2 mg/l) productstream nutrient/24 hours.

When using a medium consisting of DME/F12 with 4% fetal serum, 50 IU of insulin (recombinant human insulin) for 1 l culture medium and 1% V/V dimethyl sulfoxide, the level of expression of the target protein is 12-18 IU/ml (1.2-1.8 mg/l) productstream nutrient/24 hours.

Example 4. Cultivation of cells producing recombinant factor VIII blood clotting is a ne in the culture vessels industrial type.

For cell culture-producers and obtain productstream nutrient medium used for culture vessels Cell Factory with a total surface for cell growth=2.4 m2and displacement of the nutrient medium=8 HP Seeding density of 2.5·104CL/cm2. The growth medium has a composition: DME/F12 with 10% fetal serum; environment production: DME/F12 with 4% fetal serum, with 50 ME insuline environment products with 1% V/V dimethyl sulfoxide.

Cells were cultured at 37°C, the CO2in the gas phase=5%. After the formation of a dense monolayer cultures of cells, typically 72 hours after seeding the cells, the growth media is replaced with environment products. Environment products replace every 24 hours at the latest, of the same composition and volume.

Under these culture conditions, the expression level of the target protein is 20-28 IU/ml (2.0 and 2.8 mg/l) for 24 hours.

The selection of the obtained protein was performed using the methods described later in examples 5-8. When this was used in a unique combination of separation methods, allowing the use of this method for industrial production of a medicinal product on the basis of rhFVIII-BDD.

Example 5. The allocation of rhFVIII-BDD using katiana-exchange chromatography on SP Sepharose

The coagulation factor VIII (FVIII) is a metal-dependent protein having NESCO which are binding of different metal ions. Key ion is calcium (CA2+), which is required for the manifestation of the biological activity of FVIII. In addition to the binding of calcium, FVIII center has high affinity for copper ions (Cu2+), the binding of which stabilizes the protein structure and improves its functional properties. However, at concentrations greater than 1 mm copper ions inhibit FVIII. Thus, in the process providing all the solutions FVIII contained calcium ions (CA2+and close to equimolar protein amounts of copper ions (Cu2+).

SP-sepharose is strong katiana-exchange sorbent, linking biomolecules with a positive charge under conditions of application. Using SP sepharose in the allocation process FVIII leads to the quantitative capture of FVIII from the culture fluid, which leads to clearing of negatively charged impurities such as proteins and DNA producer strain does not bind to the sorbent, and allows you to concentrate FVIII before carrying out the following chromatographic stage.

Before applying the clarified culture fluid on SP-sepharose sorbent balanced starting buffer (20 mm imidazole, 5 mm CaCl2, 3 mm EDTA, 0.02% of Polysorbate 80, pH 7.5). After applying the sorbent was washed with the starting buffer from unbound impurities. The fraction of FVIII was suirable salt buffer (20 mm imida the ol, 5 mm CaCl2, 3 mm EDTA, 600 mm NaCl, 0.05% Polysorbate 80, 0.0001 mm CuSO4, pH 7.5). The resulting solution was subjected to anti-virus processing.

Example 6. Anti-virus processing of rhFVIII-BDD

Antivirus FVIII treatment was performed by adding to the eluate from the SP sepharose of Polysorbate 80 to 1% and three-(n-butyl)phosphate, 0.03%; the mixture was left under stirring for one hour at room temperature. When this is broken the shell of viruses containing lipids, and the infectivity of the viruses completely lost.

Example 7. The allocation of rhFVIII-BDD using affinity chromatography on CNBr IgG Sepharose.

CNBr-IgG sepharose is affine sorbent own manufacture, obtained by immobilization of monoclonal antibodies to FVIII on CNBr-activated sepharose; monoclonal antibodies were obtained by cultivation of the line mouse hybridoma. The use of affinity of the sorbent in the allocation process leads to specific quantitative binding of FVIII, resulting in the most efficient purification from residual impurities.

Column with CNBr-IgG separate pre-balanced starting buffer (20 mm imidazole, 5 mm CaCl2, 400 mm NaCl, 0.02% of Polysorbate 80, 0.0001 mm CuSO4, pH 6.8), and then applied the solution FVIII, past the anti-virus processing, and washed sorbent starting buffer. The fraction of FVIII was suirable b is from, containing 50% ethylene glycol. The obtained fraction FVIII was diluted 2-fold with dilution buffer (100 mm ammonium acetate, 30 mm NaCl, 5 mm CaCl2, 0.02% Polysorbate 80, pH 6.4) and immediately applied to the next purification step.

Example 8. The allocation of rhFVIII-BDD using anyone-exchange chromatography on Q-Sepharose

Q-Sepharose is strong anyone-exchange sorbent, linking biomolecules having a negative charge in the conditions of application and allows to separate substances according to the strength of binding. Use the Q-sepharose in the allocation process rhFVIII-BDD allows you to clear the monomer rhFVIII-BDD from related impurities (mainly degradation products), which have less affinity for the sorbent, and allows you to concentrate FVIII in the buffer ready form.

Column Q-separate pre-balanced starting buffer (100 mm ammonium acetate, 10 mm NaCl, 0.02% Tween 80, pH 6.4). After application of the diluted eluate with affine stage, the sorbent was washed with the starting buffer and then with washing buffer (100 mm ammonium acetate, 100 mm NaCl, 20 mm CaCl2, 0.02% poloxamer 407, pH of 6.4). Then the sorbent was balanced intermediate buffer (19.3 mm histidine, 100 mm NaCl, 17.5 mm sucrose, 0.02% poloxamer 407, pH 7.0), then the fraction of FVIII was suirable buffer with high concentration of calcium (19.3 mm histidine, 250 mm NaCl, 50 mm CaCl2, 17.5 mm sucrose, 0.02% Pluronic F127, pH 7.0). Received the subst is nciu FVIII in the buffer, suitable for storage or lyophilization.

Example 9. Determination of biological activity of the protein obtained.

The FVIII activity was measured by chromogenic method of analysis. The method is based on the fact that the rate of increase of optical density of the reaction mixture having the composition set clotting factors, sequentially activating each other, depends on the activity of FVIII, which is the first factor in the cascade. In the reaction mixture is made, the sample of factor VIII and the mixture of reactants consisting of activated factor IX and factor X. the Mixture incubated at 37°C and continuous stirring, and then added a chromogenic substrate for activated factor X. the Rate of change of optical density of the final reaction mixture were detected with a spectrophotometer; FVIII activity was determined relative to an international standard.

Substance FVIII obtained by the method described in the previous examples showed high specific activity of about 10,000 IU/mg.

A purified protein can be used to produce medicines.

1. Recombinant plasmid DNA RAR encoding a polypeptide with the sequence of factor VIII clotting of the person having the size of 13973 BP, physical map presented on figure 3, and consists of the following is x items:
- optimized sequence that encodes a polypeptide factor VIII blood clotting person with a deletion In the domain represented as SEQ ID N0:2,
- 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 (10094 BP), AsiSI (660 BP), BglII (2674 BP), BmtI (1085 BP), BssHII (8520 BP), BstBI (8804 BP), Eco47III (13117 BP), EcoRI (12777 BP), EcoRV (13711 P.O.), MluI (5474 P.O.), NheI (1085 po), NotI (6660 BP), PpuMI (5458 P.O.), RsrII (8638 P.O.), SbfI (5105 P.O.), SpeI (152 PP.), Tth111I (8238 P.O.), XcmI (2096 BP).

2. Cell line Cricetulus griseus CHO 2H5 - producer of recombinant factor VIII clotting of human blood, obtained by transformation of the cell line Cricetulus griseus CHO DHFR - recombinant plasmid DNA RAR according to claim 1.

3. A method of obtaining a polypeptide having the activity of factor VIII, comprising culturing the cell line according to claim 2, and secretion of the polypeptide from the culture fluid, characterized by the fact that cultivated the e, ensuring the expression of the specified polypeptide, conducted in the environment of DME/F12 containing 2-4% fetal serum, 1% DMSO, and 50 IU/l insulin.

4. The method according to claim 3, characterized in that the secretion of the polypeptide carried out using the following sequence of operations:
clarification of the culture fluid, cation-exchange chromatography on SP-Sepharose, antiviral treatment, affinity chromatography on CNBr-IgG-Sepharose, anion-exchange chromatography on Q-Sepharose.



 

Same patents:

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

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: 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

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: 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

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