Organic compounds

FIELD: biotechnologies.

SUBSTANCE: invention refers to eucariotic vector for expression of target recombinant product in a mammal cell and to its use, to a mammal cell for production of target recombinant product and to a method for its production, a method of a mammal cell selection and a method for obtaining a target recombinant product. Vector includes the first polynucleotide coding a functional folate receptor bound to a membrane as a selective marker and the second polynucleotide coding the target product that is expressed in a recombinant manner. Target product represents a pharmaceutically active, therapeutically active or diagnostic polypeptide. Functional folate receptor bound to the membrane and target product are expressed from the above expression vector. Sampling system is based on introduction of a gene of exogenic functional folate receptor bound to the membrane to a mammal cell.

EFFECT: invention allows effective selection of transformed cells and high yield of target product.

26 cl, 3 tbl, 2 ex

 

The technical field to which the invention relates.

The present invention relates to a new selection system for use in the culturing of eukaryotic cells for expression of the target recombinant product. The selection system is based on the introduction of an exogenous gene functional associated with the membrane folate receptor together with polynucleotide or the gene coding for the target product in a eukaryotic cell, and can be widely used in eukaryotic cells, the cell viability of which depends on the absorption of folic acid.

Background of invention

Selective markers and system selection are widely used in genetic engineering, recombinant DNA technology and the preparation of recombinant products, such as antibodies, hormones and nucleic acids in the culture of eukaryotic cells. The main task of such a dominant selective marker and breeding systems is the introduction of selective gene, which is under the influence of selective growth conditions provides the ability of cells to the formation of high levels of the target recombinant products.

Currently known 3 selective marker system:

(a) Glutamylcysteine system: enzyme glutamylcysteine (HS) responsible for the biosynthesis of glutamine from glutamate and ammonium. This biosynthetic reaction provides the only way of formation of glutamine in mammalian cells. Accordingly, in the absence of glutamine in the medium, the GS enzyme necessary for the survival of mammalian cells in culture. It is important that certain lines of mammalian cells, including myeloma cells of mice that do not Express the HS and thus cannot survive without exogenously added glutamine. Therefore, this cell line is an appropriate acceptor for the transfected gene GS, which in this system can function as a selective marker, which allows cells to grow in an environment that does not contain glutamine. In contrast, cell lines, for example, widely used cells Chinese hamster ovary (Cho), Express a sufficient amount of the TOS to support growth in the medium without glutamine. Therefore, when using such cells SNO as recipient cells for transvestie gene TOS, you can use specific and strong inhibitor of GS, methanesulfonamide (MCO)to inhibit endogenous GS activity to only transfectants expressing high levels of the transfected gene GS, could survive in the environment without glutamine. The main drawback of the HS system is a relatively long time (2-6 months) is electing growth to obtain cells, stable sverkhekspressiya target gene. Another disadvantage is the frequent use of a cytotoxic agent MCO to increase selective pressure. The presence of such a cytotoxic agent together with the target recombinant product (e.g., a polypeptide such as an antibody) may require additional stages of purification to remove this cytotoxic agent.

(b) selection System based on digidrofolatreduktazy/methotrexate (MT): dihydrotetrazolo (DHFR) catalyzes NADP-dependent recovery digidrofolieva acid to tetrahydrofolate acid (THF). Then THF is converted to 10-formyl-THF and 5,10-methylene-THF, which are used in the de novo biosynthesis of purines and thymidylate, respectively. SDOF is a by-product of catalytic activity timedilation (TC), which catalyzes the conversion of dUMP to dTMP in 5,10-methylene-THF-dependent reactions. Thus, DHFR plays an important role in the circulation of THF cofactors that are required for the biosynthesis of purine and pyrimidine nucleotides required for DNA replication. Therefore, cells (e.g. cells SNO), which lose DHFR gene (i.e. by targeting genomic deletions)can be used as recipients for transfection of DHFR gene in an environment that does not contain nucleot the Dov. After transfection, the cells can be exposed to gradually increasing concentrations antifolate MT, the most potent inhibitor of DHFR (Kd - 1 PM), thereby stimulating the cells to produce increased levels of DHFR. Multiple cycles of selection, the selective marker DHFR often undergoes significant gene amplification. In addition, mutant mouse DHFR with significant resistance to MT can be widely used as a dominant selective marker, which significantly enhances the acquisition of high-level MT-resistance in transfected cells. The main disadvantage of selective DHFR system/ MT is that this method uses mutagenic cytotoxic agent (MT), which can easily change the genotype of the recipient cells. In addition, it is necessary to apply specific security measures to protect people working with such agents. This often leads to MT-resistant cell populations, in which there is no expression of the target gene due to loss of functional mutations in the vector restored folate (RFC - reduced folate carrier) and/or loss of gene expression RFC, both of which disturb the uptake of MT. Another disadvantage is that the mutagenic drug MT can easily contaminate the allocated shorecrest the new target product (for example, polypeptide, e.g., antibody)contained in the medium, which requires the use of intensive, time consuming and expensive chromatographic methods to get rid of this mutagenic compounds (MT). In addition, the absence of MT in the final product must be shown by appropriate tests.

(C) selection System based on the vector restored folate: vector restored folate (RFC) is the universal expressed glycoprotein, which serves as the main carrier for the absorption restored folate, for example, 5-methyl-THF and 5-formyl-THF. However, RFC has a very weak affinity for oxidized folate, folic acid. Therefore, cells that have lost expression of RFC or have deleted genomic locus RFC, can serve as recipients for transfection selective marker gene RFC in the conditions under which restored folates such as 5-formyl-THF, are gradually withdrawn from the culture medium, thereby stimulating the cells to the expression of high levels of folate carrier. Selection system based on RFC has several shortcomings: (a) you Must use the recipient cells without RFC, in which the endogenous locus RFC "broken" or inactivated by targeted knockout or loss of functional mutations,b) RFC has an extremely weak transport affinity for folic acid and therefore this oxidized folate cannot be used for breeding, in contrast to the present system based on folate receptor, which is a unidirectional system absorption of folate and which will be described in detail below, the RFC is bidirectional folate carrier, which shows equivalent introduction and excretion of folate. This means that under conditions of low folate overexpression of RFC may be harmful to the recipient cells, which further export folate through sverhagressivnomu RFC.

The purpose of the present invention is to provide a new metabolic system of selection, which has certain advantages over the above breeding systems of the prior art. The new selection system based on the use of folate in the environment for culturing cells and for the presence of folate receptor introduced via expression vector in recombinant eukaryotic cell, designed to produce a target product. This new approach does not require the division of a gene endogenous folate receptor (FR). After the introduction of the vector, carrying at the same time selective gene FR and polynucleotide encoding target product (e.g., polypeptide), cells were cultured in selective medium containing very limited concentrations of folate. Therefore, only the cell is, which obviously sverkhekspressiya FR, can consume folate sufficient for sustained cell growth, DNA replication and cell proliferation, thus providing the opportunity for overexpression of the target product.

Oxidized folate, i.e. folic acid, as well as restored derivatives of folic acid, known as restored folate or tetrahydrofolate (THF), are a group of vitamins b-9, which are essential cofactors and/or coenzymes for biosynthesis of purines, thymidylate and certain amino acids in eukaryotic cells, especially mammalian cells. The THF cofactors especially necessary for DNA replication and, consequently, for cell proliferation. In particular, the THF cofactors act as donors one of the units in a number of linked metabolic pathways, including de novo biosynthesis of purines and thymidylate, amino acids, and the metabolism of methyl groups, including methylation of the CpG Islands DNA. In particular, cofactors THF, 10-formyl-THF (10-Cho-THF) put one unit in two key de novo reactions formyltransferase involved in de novo biosynthesis of purines. The first enzyme, glutinitdisplaymode (GARTF - glycinamide ribonucleotide transformylase)involved in the formation of the imidazole ring of purines, the lower flow reaction mediated 5-aminoimidazole-4-carboxymethylated-transformylase (AICARTF - 5-aminoimidazole-4-carboxamide ribonucleotide transformylase), leads to the formation of the precursor of purine inosine-5'-monophosphate (IMP). The latter serves as a key predecessor of AMP and GMP. Moreover, 5,10-methylene-THF (5,10-CH2-THF), is another important coenzyme THF, which functions as a key cofactor for the enzyme timedilation (TC). TS catalyzes the formation of timeintensity (dTMP from dUMP. Therefore, these folate-dependent enzymes are key mediators de novo biosynthesis of purine and siminovich nucleotides required for DNA replication. Essentially, these folate-dependent enzymes identified as targets for the action of folic acid antagonists, known as antifolates. For example, similar to the 4-by-aminotoluene acid, aminopterin and its homolog 4-amino-10-methylfolate acid, methotrexate (MT) were the first class of antimetabolites that are embedded in the clinic for therapeutic treatment of children with acute lymphoblastic leukemia (ALL). Currently antifolates are key components of the various chemotherapeutic regimes currently used for the treatment of other malignant diseases, including osteosarcoma, a cancer of mammary glands is, primary lakomy Central nervous system, horiokartsinoma and gestational USSR the neoplasia.

Unlike most prokaryotes, plants, fungi and some protozoa, which synthesize their own folate, mammalian and other eukaryotic species deprived of the biosynthesis of the cofactor THF and therefore must obtain them from exogenous sources. Currently there are three independent transport system mediating the uptake of folate and antifolate in mammalian cells:

a) the Predominant cellular transport system cofactors restored folate is a vector restored folate (RFC - reduced folate carrier). Carrier RFC (also known as representative 1 family vectors of dissolved substances 19, SLC19A1) is a ubiquitously expressed membrane glycoprotein with a molecular mass of -85 kDa, functioning as a bidirectional facilitate carrier, which mediates the upward transport of recovered folate through the exchange of organic phosphates, for example, Denisovich nucleotides, which are known to accumulate to very high intracellular levels, as well as Tiananmen - and pyrophosphate. RFC shows high affinity to THF cofactors, including leucovorin (5-formyl-THF; Kt = 1 μm), at the same time having only what about the very poor transport affinity (Kt = 200-400 μm) for folic acid, oxidized folate.

b) Another way of consumption of folate is the proton-coupled folate carrier (PCFT - proton-coupled folate transporter, also called SLC46A), which has recently been cloned. PCFT, which, apparently, is expressed independently from the RFC, functioning optimally at acidic pH values (5,5) and mediates the influx of both oxidized (for example, folic acid) and THF cofactors (i.e. restored folate), as well as a variety of hydrophilic antifolates, including MT. PCFT, which shows the optimal transport of folates and antifolates at acidic pH values (5,5), but not active at physiological pH values (7,4), plays a key role in the absorption of both folate and antifolate in the upper small intestine.

in the third method of transport, on which is based the present invention, involved in folate receptors (FR - folate receptor). FR are high-affinity folate-binding glycoproteins encoded by three different genes FRα (FR-alpha), FR(3 (FR beta) and FRγ (FR gamma). FRα (or FR-alpha) also known as folate-binding protein adults (or FDP-Adult Folate Binding Protein, and folate receptor 1 or FOLR (mice folbpl), as well as associated with ovarian cancer antigen (Ovarian cancer-Associated Antigen) or MOv 18. Receptor FRJ3 (or FR beta) also known as receptor FOLR2 (fetal) and as cocktail recipes. who and FBP/PL-1 (placental). FRγ (or FR gamma), also known as FOLR3 and as FR-G (see review Salazar M.D. and M. Ratnam, Cancer Metastasis Rev., 26(1), 2007, cc.141-152). Mature FR, which are well characterized, are homologous proteins with 70-80% amino acid identity and contain from 229 to 236 amino acids, as well as two to three N-glycosylated sites. FRα (FR-alpha) and FRp (FR beta) are membrane-bound, in particular glycosylphosphatidylinositol (GPI glycosylphosphtidylinositol)-anchored glycoproteins on the cell surface, and FRγ deprived of the GPI anchor and is secretively protein. FRα (FR-alpha) and FRp (FR beta) exhibit high affinity for folic acid (Kd = 0.1 to 1 nm), 5,10-dideuteroethanol acid (5,10 dideazatetrahydrofolic acid DDATHF; lometrexol; Ki = 0.4 to 1.3 nm, using [3H]folic acid as substrate) and BGC945 (which is an inhibitor timedilation on the basis of cyclopent[g]hintline, specifically transported exclusively by FRα (FR-alpha) and not transported by carrier recovered phosphates) (Kd=1 nm), but much lower affinity to MT (Kd>100 nm). FR-dependent absorption of folate and antifolate occurs through the classical mechanism of receptor-mediated endocytosis. Studies using knockout genes showed that the receptor FRα (FR-alpha) (also called in mice Folbp 1) very important during anego embryonic development, maternal supplementation of folate saves from in utero embryonic lethality and allows normal development of the fetus.

Currently, there is a need for a reliable, highly efficient and cost-effective selection system that overcomes one or more shortcomings of the presently known selective systems.

Brief description of the invention

The present invention relates to the eukaryotic expression vector comprising the first polynucleotide encoding functional associated with the membrane folate receptor, and the second polynucleotide encoding target product.

The present invention also relates to eukaryotic cells, in which cell viability depends on the consumption of folic acid, and in which consistently put the specified expression vector so that the cells expressed functional folate receptor encoded by this vector.

In addition, the present invention relates to a method of screening for the production of recombinant eukaryotic cells capable of stably Express high yield of the target product.

The present invention can be successfully used to obtain high yield of the target product.

Detailed description of the invention

In the present invention it has been unexpectedly discovered is, the selection system for production of recombinant eukaryotic cells capable of producing the target product may be based on limited availability of folate in the environment for culturing cells. This system can be applied widely, i.e. with respect to eukaryotic cells, whose viability depends on the consumption of folate.

The new system can be used for fast selection, screening and creating clones of eukaryotic cells, such as mammalian cells, which stably Express the ultra-high levels of recombinant products in the absence of cytotoxic drugs. In addition, and in contrast to other known systems selection, no significant needs (although sometimes valid) in the modified cells, obtained, for example, by mutation or knockout of the endogenous gene (genes). Because, for example, FRα (FR-alpha) has higher affinity to FC (KD=0.1 nm)than, for example, RFC-to-leucovorin (Kt = 1 μm), and transports folic acid into cells via a unidirectional path, the present invention provides for the use of FRα (FR-alpha) and other folate receptor as clearly superior primary metabolic selective marker, in particular, through the gradual . depletion of folate (for example, f is leeway acid) in a nutrient medium. New breeding folate is a successful strategy that is suitable for rapid and stable overexpression at a high level of target proteins in cultured mammalian cells in the absence of selection on the basis of cytotoxic drugs commonly used in various systems overexpression.

The new selection system exhibits several important advantages compared with the known in this field affordable systems selection.

1. The selection system of the present invention is very fast operating system: within four weeks of depletion of folic acid can be obtained easily derived from a population of cells or cell clone expressing the target gene. This differs from the above-mentioned system of the TOS which may require 2-6 months of selection and stabilization of the target gene.

2. The selection system of the present invention does not require genomic deletion or attenuation of endogenous genes FRα (alpha), β (beta) or γ (gamma) prior to transfection and therefore can be applied to any recipient cell, even if there is some expression of endogenous genes FR. It's main advantage is based on the fact that after transfection FRα (FR-alpha) cells can be subjected to sudden and severe depletion of folate (e.g., folic acid) Pete is positive environment. As a result, only transfected cells that Express significant amounts of the selective marker FRα (FR-alpha), can transport a sufficient amount of folate for stable DNA replication and cell proliferation. This occurs in the absence of any significant increase in the expression of the endogenous gene FRα (FR-alpha). This is in contrast to the above system DHFR/MT, in which recipient cells are often removed endogenous DHFR gene (e.g., cells Cho DG44 and Cho Dux).

C) selection System according to the present invention does not suffer from the loss severity of the selection due to the weakening of selective pressure via increased expression of other ways to absorption of folate, including increased expression of endogenous RFC. This is an important advantage due to the fact that if FRα (FR-alpha) has a high affinity for folic acid (Kd = 0.1 nm), RFC shows extremely weak affinity for folic acid (Km = 0.2-0.4 mm). On the contrary, various other selection systems in the prior art, including DHFR/MT, can be violated due to a substantial loss of intensity of selection, because the selection of MT can often be MT-resistant cells that have no or weak expression of the selective marker. Instead, the loss function RFC often leads to frequent abutment mechanism is ivoti to M initial conveyor MT. It is established that this is due to the frequent occurrence of inactivating mutations in the gene RFC or serious loss of gene expression RFC.

g) In the selection system of the present invention do not use a cytotoxic drug, agent and/or mutagenic compound, for example, the MT system DHFR or MCO in the HS system, which can change the genotype of the recipient cells, and analyzed the target gene. Rather, FR selection uses the principle of insufficiency of the vitamin in a nutrient medium.

Accordingly, one of the problems to be solved in the present invention, refers to the eukaryotic expression vector comprising the first polynucleotide encoding functional associated with the membrane folate receptor (i.e. selective marker gene), and the second polynucleotide encoding target product.

Functional associated with the membrane folate receptor of the present invention in particular is characterized as a functional associated with the membrane receptor capable of unidirectional import or admission of folate in the eukaryotic cell.

Folate in accordance with the present invention can be either oxidized folate (i.e. folic acid), restored or folate. In General, the present invention can be applied such folate, which is able to act in eukaryotic CL is woven by a function associated with the membrane folate receptor. A preferred example of oxidized folate is folic acid. A preferred example of the restored folate is 5-methyltetrahydrofolate acid, 5-formyltetrahydrofolate acid, 10-formyltetrahydrofolate acid and 5,10-methylentetrahydrofolate acid.

In a preferred embodiment of the present invention the expression vector of the present invention is capable of expression in eukaryotic cells and functional associated with the membrane folate receptor, and the target product.

Target product encoded by the second polynucleotide may be any biological product that may be generated by transcription, translation or any other event, the expression of the genetic information encoded by the second polynucleotide. In this regard, the product will be the product of the expression. For example, in a preferred embodiment of the present invention, a product chosen from the group consisting of a polypeptide, RNA and DNA. "Polypeptide" refers to a molecule comprising a polymer of amino acids linked together polypeptide bonds (link). The term "polypeptide" includes polypeptides of any length, which may be called "protein" in the case of larger molecules (including, for example, approximately alinakisa) or "peptide", in the case of smaller molecules (including, for example, 2-49 amino acids). The product can be a pharmaceutically or therapeutically effective compound or research tool used in the analyses, and other similar connection. In a particularly preferred embodiment of the present invention the product is a polypeptide, preferably a pharmaceutically or therapeutically effective polypeptide, or a research tool used in diagnostic or other tests, and other such connection. In the most preferred embodiment of the present invention, the polypeptide is an immunoglobulin molecule or an antibody, or fragment (especially functional fragment), for example, chimeric, or partially or fully humanized antibody. This antibody can provide a diagnostic antibody, or a pharmaceutically or therapeutically effective antibody. Typically, the target product can be heterologous in relation to the eukaryotic host cell used for expression, which means that prior to transfection of a host cell does not produce the target product in a natural way or endogenous. More precisely, to obtain a product or expression of the target product, it is necessary to enter in the eukaryotic cell host polynuclear is d, encoding the desired product, in particular by transfection with the expression vector of the present invention.

The vector of the present invention can be presented in linear form or, preferably, in an annular shape, for example, in the form of plasmids.

The vectors used for expression of the target polynucleotide, usually contain elements that control transcription, suitable for transcription, such as promoters, enhancers, polyadenylation signals, transcriptional signals a pause or termination. If the target product is a protein, the vector typically includes appropriate controls broadcast, for example, a 5' untranslated region, leading to the 5' cap structures, suitable for recruitment of ribosomes, and stop codons to stop the process of translation. In particular, both polynucleotide, polynucleotide that acts as a selective marker gene, and polynucleotide encoding the target product can be transcribed under the control of the transcription present in the respective promoters. The resulting transcripts of both genes, gene selective marker and the gene of the target product, contain functional elements of the broadcast, which facilitate receiving significant levels of protein expression (i.e. broadcast).

Thus, preference is sustained fashion option of implementing the present invention relates to the expression vector of the present invention, in which the first polynucleotide and second polynucleotide are under the control of separate promoters for transcription. In General, a suitable promoter capable of promoting expression, particularly transcription, the first and/or second polynucleotide in eukaryotic cells. In a preferred embodiment of the present invention, a separate transcriptional promoters are identical. In another preferred embodiment of the present invention separate transcriptional promoters are different. Preferably, the transcriptional promoters are selected from the group consisting of the SV40 promoter, the murine promoter of cytomegalovirus (CMV)promoter, EF1-alpha, RSV promoter, promoter BROAD3, promoter rosa 26 promoter pCEFL and promoter of β-actin. In a preferred embodiment of the present invention the promoter controlling transcription of the first polynucleotide and/or the second polynucleotide is a CMV promoter or, most preferably, the SV40 promoter. In a particularly preferred embodiment of the present invention the promoter controlling transcription of the first polynucleotide is the SV40 promoter.

In another preferred embodiment of the present invention the expression vector of the present invention, the first polynucleotide and second polynucleotide are under the control of a common promoter transcription. Preferably such a promoter transcription is selected from the group consisting of the SV40 promoter, CMV promoter, RSV promoter, promoter BROAD3, mouse promoter rosa 26 promoter pCEFL and promoter of β-actin. In another preferred embodiment of the present invention into a vector expression of the General transcriptional promoter is the SV40 promoter. In yet another preferred embodiment of the present invention the expression vector having such a General promoter transcription includes the IRES element, functionally localized between the first polynucleotide and second polynucleotide.

Associated with the membrane folate receptor, which is introduced into the eukaryotic cell host by means of the expression vector used in accordance with the present invention, can be obtained from any species, if it is to operate in accordance with the present invention, that is compatible with the eukaryotic cell. Preferably use folate receptor, obtained from a species of mammal, for example obtained from a rodent, or, most preferably, folate receptor human. In General, the folate receptor is introduced into the eukaryotic cell host and used as a selective Marker may be homologous or heterologous to the endogenous folate receptor to etki host. When he homologous, it is derived from the same species as the cell-master, and he may, for example, be identical to the endogenous folate receptor of the host cell. When he is heterologous, it is derived from another species other than the species from which the received cells-owners, and thus it may differ from the endogenous folate receptor of the host cell. Usually introduced in folate receptor, used as a selective marker may be heterologous to the cell master. For example, folate receptor, obtained from a person, can be used as a selective marker for host cells of rodent, for example, cells SNO.

Preferably, the functional associated with the membrane folate receptor encoded by the first nucleotide of the expression vector of the present invention, selected from the group consisting of folate receptor FRα (FR-alpha), FRP (FR beta) and their functional mutants. Functional mutant includes a derived folate receptor, which operates a physiological way, i.e. can be absorbed eukaryotic cell and maintain the viability of the cells through the folate metabolism of the cell. For example, the mutant form of folate receptor may include one or more amino acid mutations on the type of substitution, deletions and/or additions, as well as helices the second derivative, moreover, the chemical part, similar to the polymer, for example, patterns of polyethylene glycol (PEG)attached to the folate receptor. Preferably, folate receptor, encoded by the first polynucleotide is folate receptor alpha (hFRα) human folate receptor beta (hFRP) of a person or their functional mutant. The most preferred folate receptor alpha (hFRα) person, preferably having the following amino acid sequence (SEQ ID NO 1, a single-letter code shown in the direction from N-Terminus to the C-end):

MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS

Another preferred implementation of the present invention relates to folate receptor beta (hFRP) a person having the following amino acid sequence (SEQ ID NO 2, a single-letter code shown in the direction from N-Terminus to the C-end):

MVWKWMPLLLLLVCVATMCSAQDRTDLLNVCMDAKHHKTKPGPEDKLHDQCSPWKKNACCTASTSQELHKDTSRLYNFNWDHCGKMEPACKRHFIQDTCLYECSPNLGPWIQQVNQTWRKERFLDVPLCKEDCQRWWEDCHTSHTCKSNWHRGWDWTSGVNKCPAGALCRTFESYFPTPAALCEGLWSHSYKVSNYSRGSGRCIQMWFDSAQGNPNEEVARFYAAAMHVNAGEMLHGTGGLLLSLALMLQLWLLG

In another embodiment, the present invention relates to folate receptor, which in its natural environment is not associated with the membrane. This is not associated with the membrane receptor can be modified to bind to the membrane, for example, providing a hybrid protein between not associated with the membrane folate receptor and the transmembrane region of another polypeptide. There might also be other mutant forms that are readily accessible to qualified specialists in this field. Preferred examples in this regard can be based on soluble folate-receptor gamma (FRγ), preferably soluble folate-receptor gamma (FRγ) person. In the most preferred embodiment, soluble folate receptor gamma (FRγ) may have the following amino acid sequence (SEQ ID NO 3, a single-letter code shown in the direction from N-Terminus to the C-end):

MDMAWQMMQL LLLALVTAAG SAQPRSARAR TDLLNVCMNA KHHKTQPSPE DELYGQCSPW KKNACCTAST SQELHKDTSR LYNFNWDHCG KMEPTCKRHF IQDSCLYECS PNLGPWIRQV NQSWRKERIL NVPLCKEDCE RWWEDCRTSY TCKSNWHKGW NWTSGINECP AGALCSTFES YFPTPAALCE GLWSHSFKVS NYSRGSGRCI QMWFDSAQGN PNEEVAKFYA AAMNAGAPSR GIIDS

which can then be genetically modified or can be obtained its derivative to create a functional associated with the membrane folate receptor, is able to consume folate in the context of the present invention.

Another object of the present invention the expression vector of the present invention may optionally include one or more other polynucleotides encoding one or more selective markers. Accordingly, preferable is the version of the implementation of the present invention for its optimum performance, it is possible to apply jointly selected with the use of folate system according to the present invention together with one or more different systems selection (for example, neomycin/0418).

Another object of the present invention relates to eukaryotic cells, cell viability, which depends on the absorption of folate, and which stably introduced the first polynucleotide, localized on the expression vector and encodes a functional associated with the membrane folate receptor, and the second polynucleotide, localized on the expression vector, and encoding the desired product, and the first polynucleotide and second polynucleotide localized on the same expression vector or on different vectors of expression. In a preferred embodiment of the present invention the functional associated with the membrane folate receptor and target product expressed eukaryotic cell.

In addition to the functional associated with the membrane folate receptor introduced into the cell line with the expression vector, a eukaryotic cell in accordance with the present invention may include at least one endogenous unidirectional functional folate transport system, in particular one or more functional endogenous membrane-bound folate receptors. The advantage of the present invention is that the method of selection presented in this description below, can use the sterile even in the presence of such endogenous unidirectional functional folate transport system, where is stored this endogenous system. Accordingly, another preferred embodiment of the invention relates to a eukaryotic cell of the present invention, comprising at least one endogenous unidirectional functional folate transport system in which such endogenous unidirectional functional folate transport system preferably includes at least one endogenous functional associated with the membrane folate receptor. In a preferred embodiment of the present invention endogenous functional associated with the membrane folate receptor selected from the group consisting of folate receptor alpha (FRα) and folate receptor beta (FRP).

Another preferred implementation of the present invention relates to a eukaryotic cell according to the present invention, in which endogenous unidirectional functional folate transport system, for example, includes at least one endogenous functional associated with the membrane folate receptor, does not have full activity, i.e. is weakened. This weakening can be provided, for example, by means of any type of mutagenesis considered endogenous folate transport system, for example, endogenous funktsionalizirovannogo with membrane folate receptor, for example, by point mutation, gene destruction, etc. Weakening may be partial or complete. In the latter case, the eukaryotic cell according to the present invention does not include endogenous unidirectional functional folate transport system, for example, endogenous functional associated with the membrane folate receptor. Accordingly, in a preferred embodiment, the present invention relates to a eukaryotic cell that stably introduced the expression vector of the present invention, and which has lost total activity, at one endogenous functional associated with the membrane folate receptor.

As for the expression vector introduced into the specified cell host, you can use any expression vector of the present invention, including its preferred options for implementation, according to the present description. In a preferred embodiment of the present invention in a eukaryotic cell of the present invention the first polynucleotide encoding functional associated with the membrane folate receptor, and the second polynucleotide encoding target product, localized on the same expression vector. Preferably, such an expression vector is an expression vector according to the present invented the Yu, i.e. described in this invention.

Eukaryotic cell in accordance with the present invention is preferably selected from the group consisting of mammalian cells, insect cells, plant cells and fungal cells. With regard to fungal cells and plant cells, which are normally prototrophic in respect of folate (i.e Autonomous cells can synthesize their own folate, essential for cell viability, i.e. cell growth and proliferation). The present invention covers such fungal and plant cells, which can become auxotrophy in relation to folate. This may occur, for example, through genetic manipulation, i.e. cells become unable to synthesize adequate amounts of folate required for cell viability. For example, the ability of such fungal or plant cells endogenous to biosynthesizing folate, for example, by an appropriate metabolic pathway, is inactivated, for example by gene loss or gene silencing of the corresponding gene targets, or by inhibiting key enzymes etc.

In a preferred embodiment of the present invention the eukaryotic cell is a cell of a mammal. Preferably the such cell of a mammal selected from the group consisting of rodent cells, human cells and cells of the monkey. A particularly preferred cell is a rodent, which is preferably selected from the group consisting of cells, Cho cells, KSS, NS0 cells, mouse fibroblast cells T cells and SP2/0. A particularly preferred cell is a rodent cell is SNO. Preferred cell, which is preferably selected from the group consisting of cells NC, cells MCF-7, cells Rex and HeLa cells. Also preferred the monkey cage, which is preferably selected from the group consisting of cells COS-1 cells, COS-7 and Vero cells.

In another embodiment, the present invention relates to a method for producing eukaryotic cells according to the present invention, which includes obtaining eukaryotic cells, in which cell viability is dependent on dietary folate, and the introduction of the first polynucleotide, localized on the expression vector and the coding of the functional associated with the membrane folate receptor, and the second polynucleotide, localized on the expression vector and the coding target product, and the first polynucleotide and second polynucleotide localized on the same expression vector, which, in the preferred embodiment, an expression vector in accordance with the present image is taneem, i.e. described in this invention.

Another object of the present invention relates to a method of selection in eukaryotic cells capable of stably Express the target product encoded by the expression vector introduced into the cell, including:

(i) obtaining a variety of eukaryotic cells, in which cell viability is dependent on dietary folate, and entered the first polynucleotide, localized on the expression vector and encodes a functional associated with the membrane folate receptor, and the second polynucleotide, localized on the expression vector and the encoding target product, and the first polynucleotide and second polynucleotide localized on the same expression vector or on different vectors, expression

(ii) culturing the specified variety of eukaryotic cells in cell culture medium with a limited concentration of folate, thereby obtaining a eukaryotic cell, which is achieved stable expression of interest product. In principle, this folate may be oxidized or the reduced folate folate. Preferred oxidized folate, which, in particular, presents folic cell.

As for the limited amount of folate, a qualified expert in this field can moustache is anovice the corresponding concentration in the environment in accordance with the needs of the host cell and severity criteria, which will be applied. When used as folate, folic acid, for example, with the host-cell SNO, the corresponding concentration of folic acid in cell culture medium for a strict selection process may be approximately 100 nm or less, preferably about 30 nm or less, or about 10 nm or less. For example, the corresponding concentration of folic acid can have any value in the range of 0.001 nm to 100 nm, preferably in the range of 0.01 nm - 100 nm, more preferably in the range of 0.1 nm - 100 nm or in the range 1 nm - 100 nm. Also preferred range of 0.001 gr - 30 nm, the range of 0.01 nm to 30 nm, in a range of 0.1 nm to 30 nm, range 1 nm - 30 nm, or a range of 3 nm - 10 nm. For example, suitable for the selection of the concentration of folic acid in the cell culture medium may be 1 nm, 3 nm, 10 nm or 30 nm.

In the case when in the process of selection will be used restored folate such as leucovorin, its concentration by a qualified specialist in this field can also be set in accordance with the needs of the host cell and severity of the conditions of selection, which will be used. This concentration of leucovorin in the cell culture medium may be for example in the range of 0.2-2 nm for accurate selection process.

In a preferred is ariante implementation of the present invention, the method of selection also includes the identification and isolation of eukaryotic cells, which is achieved stable expression of the target product.

In the most preferred embodiment of the method of selection of many eukaryotic cells are eukaryotic cells according to the present invention, i.e. described in this invention.

In preferred embodiments, the implementation object of the present invention described in the present description, also presents preferred embodiments of in particular, in respect of eukaryotic cells and expression vector.

Another variant implementation of the present invention relates to a method of obtaining the target product, including:

(i) carrying out the method of selection of the present invention, i.e. in accordance with the present description,

(ii) the selection of the target product from the specified cell culture medium or from the specified cell.

In addition, the object of preferred embodiments of the present invention described in the present description, in particular in respect of eukaryotic cells and expression vector.

Target product, for example the polypeptide produced in accordance with the present invention, it is possible to allocate, to further purify and isolate using known in the field of methods. For example, the polypeptide can be isolated from the feeder is th environment traditional methods including, but not limited to, centrifugation, filtration, ultrafiltration, extraction or precipitation. Cleaning can be various well-known in this field of ways, including, but not limited to, chromatography (e.g. ion exchange, affinity, hydrophobic, chromatofocusing and exclusion), electrophoretic methods (e.g., preparative isoelectric focusing), differential dissolution (for example, precipitation with ammonium sulfate) or extraction.

Another object of the present invention relates to the use of the functional associated with the membrane folate receptor as a selective marker for selection in eukaryotic cells, in which cell viability is dependent on dietary folate, and which is capable of stably Express the target product. Under the preferred alternative implementation of this application, folate receptor selected from the group consisting of folate receptor alpha (FRα), folate receptor beta (FRβ) and their functional mutant. Preferably, folate receptors used in the specified object of the present invention, are relevant folate receptors with the preferred folic acid receptor alpha (FRα) person. In the present description presents other prepost the tion embodiments of the specified object of the present invention, in particular, in respect of eukaryotic cells and expression vector.

The full content of texts and documents referred to in the present invention included in the present description as a reference.

The following examples are intended to illustrate the present invention without any limitation of its scope. In particular, the examples refer to the preferred options of the implementation of the present invention.

Examples

All materials used in this invention, for example, reagents, familiar qualified, commercially available and may be used in accordance with the manufacturers ' instructions.

Example 1. High level expression of recombinant antibodies using selective system based on folate receptor Example 1.1. The expression vectors

To study the effectiveness of selection hFR (hFRα)-transfected cells under conditions of limited concentrations of folate, i.e. folic acid in the culture medium, creating plasmid vector (i.e. the test vector suitable for expression in eukaryotic cells, particularly in cells of SSC containing both (i) the expression cassette, which includes polynucleotide encoding heavy and light chains secreted recombinant human antibodies type IgGl, and (ii) about the practical expression cassette, which includes polynucleotide encoding the folic acid receptor alpha (hFRα) as selective gene marker. The expression of the folic acid receptor alpha (hFRα) the person is under control of the SV40 promoter and standard (SV40) polyadenylation signal. Expression of recombinant antibodies is under control of the CMV promoter and the standard (SV40) polyadenylation signal. As a control (i.e. a control vector), use a close expression vector encoding the same antibody that has lost the expression cassette hFR (hFRα), but containing as a selective marker gene neomycinphosphotransferase.

Example 1.2. Cells and culturing conditions

The ovarian cells of Chinese hamsters received from strain Cho-K1, support in terms of suspension culture in the corresponding chemically defined nutrient medium containing 2,3 μm folic acid.

To examine the dependence of the survival rate of cells from folic acid, experiment with starvation on folic acid, using the concentration of folic acid in the range from 2300 nm to 0.1 nm. Cells cultivated in this environment, and analyze cell viability to determine the percentage of surviving cells. Table 1 summarizes the results obtained with the above-mentioned cell line Cho-K1.

Table 1The survival of cells SNO at various concentrations of folic acidThe concentration of FC [nm]The survival rate0,12,0812,4532102,7306,810055,530088,61000100,82300100

The results show that for this specific cell line-hosts concentration of folic acid below 100 nm, preferably below 30 nm, applicable to create a significant selective pressure for selection based on the folic acid receptor stably transfected cells.

Example 1.3. Transfection and selection

Cells transferout by electroporation or the test vector containing the expression cassette hFR (hFRα), or control the major vector without hFR (hFRα). Then, the transfected cells are grown in suspension culture conditions in 125 ml cachelock flasks in medium with the addition of appropriate concentrations of glutamine and 2.3 μm folic acid. Forty-eight hours after transfection, the cells are transferred into a medium containing a limited amount of folic acid, namely, 10 nm or 1 nm folic acid, to initiate sampling in accordance with the present invention in a 24-hole tablets for samples transfected with the test vector. Additionally, selected cells, transfected with a control plasmid, by adding a selective agent, namely 0.8 mg/ml G418 to the medium containing the 2.3 μm folic acid (i.e. control 1), or cultured in the absence of any selection (i.e. control 2). Cells that successfully obtained as a result of this scheme selection, transferred into 6-hole tablets and then multiply in katalozhnyh flasks to analyze their levels of production of antibodies.

Example 1.4: Analysis of antibody production

For analysis of expression levels of the antibody from transfected and deprived of folic acid cell populations prepared in katalozhnyh flasks superdense cell culture. Cells were seeded at a density of 2×105cells/ml in medium containing 2,3 μm folic acid, and incubated in the conditions suspendiruemye culture (i.e. when shaken). the 14 th day collect supernatant of cell culture and analyze the levels of antibodies, using HPLC method with protein A, i.e. an affine type of treatment. The IgG molecule is specifically bound to the column, mainly through their Fc part, but other proteins pass through the column without interacting with the media. Captured IgG proteins elute from the column at low pH values, determines the amount by measuring UV absorption and if necessary, conduct further purification and selection.

Example 1.5: Results

The goal of this approach is the confirmation of the correctness of the concept lies in the fact that folate gene, in particular a gene ProLife (hFRα), may serve as a selective marker in terms of depletion of folate, thus allowing to select cells that are simultaneously sverkhekspressiya target product, for example, a monoclonal antibody. As a control, also use a vector containing as a selective marker gene of resistance to neomycin. After transfection, the cells are subjected to a stringent selection process by a sudden reduction of the concentrations of folic acid in the environment from 2.3 μm to 10 nm or 1Nm. Cells transfected with a plasmid bearing a receptor for folic acid, are easily reduced under conditions of folate deficiency and can be further propagated in selective medium. On the contrary, in the case of the control vector concentration of folic keys which the notes are left unchanged, but use either selective pressure with G418, or do not apply selective pressure. In selected cell populations then analyze the production of antibodies, using excessively grown (i.e. superdense) suspension (i.e. shake the flasks culture in a medium containing 2,3 μm folic acid. Then on the 14th day determine the concentration of antibody in the culture medium. Below in the table. 14 shows that cells transfected with a plasmid containing the receptor for folic acid, and selected by reducing the content of folic acid, sverkhekspressiya recombinant antibody. The number of antibodies generated by these populations of transfected cells is higher relative to cells transfected with control vector and selected with the antibiotic G418. As for the other controls when not in use selective pressure, get cells that do not form antibodies. These data support the concept that this approach is based on receptor gene folic acid as a standalone dominant metabolic selective marker, you can easily apply for quick detection of cells, sverkhekspressiya target recombinant product.

Table 2
(C Folic acid: concentration of folic acid in the medium for selection; CG418: concentration G418in the environment for selection; Withmb: concentration of secreted antibodies in the environment crops with excessive growth)
VectorWithfolic acidCG4I8
(nm)(mg/ml)(mg/l)
The test-vector10no25
(hFR(hFRα))1no24
Control vector (neomycin)23000,88
2300no0

Example 2. Increased levels of production of recombinant antibodies as a function of the decrease in the concentration of folic acid in a nutrient medium

Example 2.1. The expression vector of

Receive a plasmid vector (i.e., the test-vector)as described in example 1.1.

Example 2.2. Cell and growth conditions

Cells Chinese hamster ovary that are derived strain Cho-K1, support in terms of monolayer culture in a chemically defined culture medium RPMI-1640 containing 2,3 μm folic acid. Cells deprived of activity vector RFC, as described in other sources (Y.G. Assaraf and R.T. Schimke, Proc. Natl. Acad. Sci. USA, 84, 1987, cc. 7154-7158; L. Rothem and others, Mol. Pharmacol., 68, cc. 616-624). Such RFC-defective cells use to avoid potential workaround starvation on folic acid by means of such extension system carrier in this example.

Example 2.3. Transfection and selection

RFC-deficient cells C15 transferout test vector by electroporation. Forty-eight hours after transfection, the cells multiply in an environment that does not contain folic acid, enriched with 30 nm of folic acid to stimulate the expression of a selective marker, and recombinant antibodies, and subsequently subjected to cloning by dilution method. Cells are diluted to a final density of 5 cells/ml and the suspension contribute 100 μl/well in 96-well plates (i.e. 0.5 cells/well). Then the clones support in the medium containing 0.25 nm folic acid and 500 μg/ml G418. To ensure optimum process selection, use of joint selection using folate system according to the present invention instead of the e with an additional selection system (i.e. neomycin/0418). Then the clones with the highest levels develop antibodies are grown at low concentrations of folic acid (i.e. 1200 peak, 600 peak and 60 peak) for further support and adapting overexpression antibodies. This is confirmed by further analysis of the expression of antibodies in different clones.

Example 2.4: Analysis of antibodies

Analysis of the production of antibodies is conducted according to the principle described above in example 1.4. The concentration of secreted antibodies are monitored using enzyme-linked immunosorbent assay ELISA according to the following scheme: Maxisorp microplates coated with antibody against human IgG. After blocking buffer containing bovine serum albumin (BSA), and washed several times with add multiple dilution secreted antibodies. Then add the second antibody conjugated with peroxidase, consisting of goat antibodies against human IgG and peroxidase. In conclusion, type-specific peroxidase colorimetric substrate, after which in each well spectrophotometrically determine the resulting concentration of the dye and then compare with standard concentrations of known concentrations of IgG.

2.5. Results

According to the results, shown below in table. 2, the production levels of recombinant antibodies clearly q is leraut levels lack of folic acid. Thus, the levels of production of antibodies increase with the decrease in the concentration of folic acid. In addition, these results confirm the concept that the gene Irjala (hFRα) is a selective marker, which can be used for the overexpression of recombinant proteins under conditions of low folate.

Table 3
(CFolic acid: concentration of folic acid in the environment; mAb'- the concentration of secreted antibodies in the environment)
Withfolic acid(PM)mb (UG/L)
60216±30
600138±15
120019±8

1. The eukaryotic expression vector for recombinant expression of a target product in a cell of a mammal, comprising the first polynucleotide encoding functional associated with the membrane folate receptor as a selective marker, and the second polynucleotide encoding target product, which recombinante is expressed, where the target product is pharmaceutically active, therapeutically act the main or diagnostic polypeptide, and where the functional associated with the membrane folate receptor and target product expressed from the expression vector.

2. The expression vector according to claim 1, in which the first polynucleotide and second polynucleotide are under the control of separate promoters of transcription.

3. The expression vector according to claim 2, in which the promoters of transcription are the same.

4. The expression vector according to claim 2, in which the promoters of transcription of different.

5. The expression vector according to any one of claim 2 to 4, in which the promoter controlling transcription of the first polynucleotide is the SV40 promoter.

6. The expression vector according to claim 1, in which the first polynucleotide and second polynucleotide are under the control of a common promoter transcription.

7. The expression vector according to claim 6, in which common promoter transcription is the SV40 promoter.

8. The expression vector according to claim 6, wherein said vector comprises an IRES element, functionally localized between the first polynucleotide and second polynucleotide.

9. The expression vector according to claim 1, in which the functional associated with the membrane folate receptor encoded by the first polynucleotide selected from the group consisting of folate receptor alpha (FRα), folate receptor beta (FRβ) and their functional mutant.

10. The expression vector according to claim 9, in which the functional associated with membranes is th folate receptor, the encoded first polynucleotide is folate receptor alpha (hFRα) person.

11. The cell of a mammal to produce the target recombinant product, and cell viability specified cells depends on the consumption of folate, and which stably introduced the first polynucleotide, localized on the expression vector and encodes a functional associated with the membrane folate receptor, and the second polynucleotide, localized on the expression vector and the encoding target product, in which the first polynucleotide and second polynucleotide localized on the same expression vector or on separate expression vectors, and where the functional associated with the membrane folate receptor and target product recombinante expressed by the cell of a mammal, and where the target product is a pharmaceutically active, therapeutically active or diagnostic polypeptide.

12. The cell of a mammal according to claim 11, with the specified cell loses the full action of at least one endogenous functional associated with the membrane folate receptor.

13. The cell of a mammal according to claim 11 or 12, in which the first polynucleotide encoding functional associated with the membrane folate receptor, and the specified second polynucleotide encoding target PR the product, localized on the same expression vector.

14. The cell of a mammal according to claim 11, in which the expression vector is a vector that is described in one of claims 1 to 10.

15. The cell of a mammal according to claim 11, in which the specified cell of a mammal is a rodent cage.

16. The cell of a mammal according to § 15, in which the cell is a rodent cell is SNO.

17. The method of obtaining cells of a mammal according to any one of § § 11-16, including the production of mammalian cells, cell viability, which depends on consumption of folate, and the introduction of the first polynucleotide, localized on the expression vector and the coding of the functional associated with the membrane folate receptor, and the second polynucleotide, localized on the expression vector and the coding target product, in which the first polynucleotide and second polynucleotide localized on the same expression vector or on separate vectors expression.

18. The method according to 17, in which the first polynucleotide and second polynucleotide localized on the same expression vector.

19. The method according to p, in which the expression vector is the vector according to any one of claims 1 to 10.

20. The method of selection of mammalian cells capable of stably Express the target product encoded by the expression vector, which is introduced into the cell, including:
i) obtaining a variety of mammalian cells, cell viability of which depends on the consumption of folate, and entered the first polynucleotide, localized on the expression vector and encodes a functional associated with the membrane folate receptor, and the second polynucleotide, localized on the expression vector and the encoding target product, and the first polynucleotide and second polynucleotide localized on the same expression vector or on separate vectors, expression,
(ii) culturing the specified variety of mammalian cells in a medium for culturing cells, has limited the concentration of folate, thereby obtaining a cell of a mammal, which is achieved stable expression of the target product.

21. The method according to claim 20, additionally including the identification and isolation of mammalian cells, which is achieved stable expression of interest product.

22. The method according to claim 20 or 21, in which many mammalian cells include mammalian cells by any of § § 11-16.

23. The method of obtaining the target recombinant product, including:
(i) implementation of the method of selection according to any one of p-22, and
(ii) isolation of the target product from the specified cell culture medium or from the specified cell.

24. Application of the functional associated with the membrane folate receptor, the cat is which is injected through the expression vector, as a selective marker for selection of mammalian cells, the viability of which depends on consumption of folate, and which can stably Express the target recombinant product, where the target product is a pharmaceutically active, therapeutically active or diagnostic polypeptide.

25. The application of paragraph 24, and folate receptor selected from the group consisting of folate receptor alpha (FRα), folate receptor beta (FRβ) and their functional mutant.

26. Use A.25, and folate receptor is a human folate receptor alpha (hFRα) person.



 

Same patents:

FIELD: biotechnologies.

SUBSTANCE: expression vector includes: (a) replication origin OriP obtained from Epstein-Barr virus (EBV), where replication origin contains: 1) symmetry element of the second order (DS); and 2) duplication section (FR) that contains fixation point EBNA; (b) replication origin SV40; (c) insertion section for inserting a gene of concern; (d) promoter EF-1b functionally bound to the insertion section; (e) poly-A signal; (f) bacterial replication origin; (g) selected marker; and unnecessarily containing (h) sequence of nucleic acid, which codes constant area of heavy or light chain of antibody, which is functionally bound to the insertion section. With that, replication origin OriP is bound to an initiation factor of replication EBNA 1, which acts from outside and is not coded with an expression vector.

EFFECT: use of an expression vector in an extracted host cell, a set and a method for obtaining recombinant protein provides production of abundant protein expression.

26 cl, 25 dwg, 3 tbl, 4 ex

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

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: chemistry.

SUBSTANCE: invention relates to a peptide capable of binding with scurfin and inhibiting biological activity of scurfin, which is selected from a peptide consisting of an amino acid sequence Arg-Asp-Phe-Gln-Ser-Phe-Arg-Lys-Met- Trp-Pro-Phe-Phe-X, where X is absent or X is present and represents X14 or X14-X15, where X14 and X15 independently denote an amino acid, a version of said peptide and a pharmaceutically acceptable salt thereof. The invention also discloses a fused protein and a pharmaceutical composition, which involves use of said peptide and fused protein, as well as use thereof to produce and treat pathologies which require transient regulation or inhibition of immunosuppressive activity of regulatory T lymphocytes, such as a neoplastic disease or infectious disease. The invention also relates to a method of producing said peptide and fused protein, including a protein or peptide coding nucleic acid, a DNA construct, an expression vector and a host cell.

EFFECT: invention provides effective treatment of infectious and neoplastic diseases which require transient regulation or inhibition of immunosuppressive activity of regulatory T lymphocytes.

26 cl, 10 dwg, 5 ex

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

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: invention represents recombinant BMPRIA-CBD plasmid, and E.coli strain transformed with that plasmid. The invention also refers to recombinant BMPRIA-CBD protein that is used for production of BMP-2 protein.

EFFECT: invention allows producing chromatographic clean fractions of biologically active dimeric form of BMP-2 protein, which can be used as osteoinductive components of osteoplastic materials of new generation.

4 cl, 2 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents recombinant BMPRIB-CBD plasmid, and E.coli strain transformed with that plasmid. The invention also refers to recombinant BMPRIB-CBD protein that is used for production of BMP-7 protein.

EFFECT: invention allows producing chromatographic clean fractions of biologically active dimeric form of BMP-7 protein, which can be used as osteoinductive components of osteoplastic materials of new generation.

4 cl, 2 dwg, 6 ex

FIELD: biotechnologies.

SUBSTANCE: versions of an antibody or its fragment, which are specific in relation to β-amyloid protein, are proposed. Each version is characterised by the fact that it includes H- and L-chains, or areas VH and VL, each of which contains three corresponding CDR. The following is described: polypeptide VL, polypeptide VH, as well as coding nucleic acid, expression vector containing it and a cell carrying the vector, which are used for obtaining an antibody or its functional fragment. The following is proposed; a test kit, versions of pharmaceutical composition, a mixture to be used as a medicine based on the antibody or its functional fragment. Versions of the method used for production of an antibody are described: using a cell, nucleic acid or a vector. A composite preparation method, as well as an in vitro amyloid disease diagnostics method, a method for determination of a degree of loading with in vitro amyloidogenic patches, a method for curing or relief of actions of amyloid disease, which use an antibody or its functional fragment, are described. Inventions can be used in therapy and diagnostics of Alzheimer disease and other enlisted amyloid diseases.

EFFECT: proposed inventions provide new antibodies that bind the epitope contained in the area of 12-23 protein αβ1-42; with that, residues 15-20 have a fundamental importance.

44 cl, 18 dwg, 9 tbl, 16 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: biotechnologies.

SUBSTANCE: invention proposes a method for obtaining an expression vector coding an adapted recombinase. The above adapted recombinase recombines asymmetric sections-targets in LTR of proviral DNA of retrovirus built into a genome of a host cell and applicable as means for cutout of provirus from genome of the host cell.

EFFECT: optimisation of curing of retroviral infection with individual in vitro and use of adapted recombinases for obtaining pharmaceutical compositions for reduction of virus load with the individual infected with retrovirus.

21 cl, 11 dwg, 1 tbl, 4 ex

Up!