RU2448160C1 - RECOMBINANT PLASMID DNA pAP271 CODING HUMAN BLOOD COAGULABILITY FACTOR VII POLYPEPTIDE AND Mesocricetus auratus BHK 21 k. 13 (2H7) CELL LINE PRODUCER OF RECOMBINANT HUMAN BLOOD COAGULABILITY FACTOR VII - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: there is constructed a recombinant plasmid DNA pAP271 containing a rhFVII protein gene, a MAR matrix attachment region of an avian lysozyme gene, CMV virus transcription amplifier, a promoter of translational factor of human EF-1α elongation, an internal site of translational initiation of encephalomyocarditis IRES virus, a mouse DHFR gene, a SV40 virus polyadenylation signal, a cartridge comprising all elements required for aminoglycoside-3'-phosphotransferase (APH) gene expression, a cartridge for expression in bacterial cells of β-lactamase gene, as well as unique recognition sites of the following restriction endonucleases: Agel (850 base pairs), BbvCI (1657 base pairs), BmgBI (4202 base pairs), BssHII (6672 base pairs), Eco47III (11269 base pairs), EcoRI (10929 base pairs), EcoRV (11863 base pairs), Fsel (1455 base pairs), NotI (4812 base pairs), RsrII (6790 base pairs), Sbfl (2330 base pairs), Sfil (6027 base pairs), Tthllll (6390 base pairs), Xcml (2404 base pairs).

EFFECT: presented invention provides producing stably high-yield recombinant human blood coagulability factor VII.

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Description

The invention relates to biotechnology, in particular to genetic engineering, and can be used to produce recombinant activated human coagulation factor VIIa (rhFVIIa). The invention is a recombinant plasmid DNA pAP271 encoding a polypeptide with a sequence of a recombinant human blood coagulation factor VII (rhFVII), and a producer strain based on the newborn hamster kidney cell line Mesocricetus auratus BHK21 k.13 (2H7) containing the indicated plasmid recombinant DNA and human coagulation factor VII.

Coagulation factor VII of a person is a glycoprotein, the molecular weight of which is about 55 kDa, which is a component of blood plasma. The hFVII protein circulates in the bloodstream in an inactive single-chain form and is a zymogen. It can undergo limited proteolysis using various proteases of plasma components, for example, factor Xa, XIIa, IXa or thrombin, or autoproteolysis. In this case, the cleavage of the polypeptide chain between Arg152 and Ile153 occurs and the formation of two polypeptide chains, called light and heavy, held together in a single complex using a disulfide bond. This process is called activation of factor VII. The main physiological activator of factor VII is factor Xa. Thus obtained FVIIa is further involved in the process of blood coagulation, leading to the formation of a fibrin clot.

Like some other plasma proteins involved in the process of hemostasis, the activity of factor VII is vitamin K-dependent. Vitamin K is necessary for gamma-carboxylation of 10 glutamine residues arranged in a cluster at the N-terminus of the protein sequence, which provides the metal-dependent interaction of FVII with phospholipids necessary to activate coagulation.

rhFVIIa has two N- and O-glycosylation sites. O-glycosylation sites are located on serine residues 52 and 60 and are necessary for interaction with epidermal growth factor (EGF). N-glycosylation sites are represented by two residues Asn145 and Asn322. O-glycosylation sites contain from 1 to 4 monosaccharide residues, N-glycosylation sites are more complex in nature and represent bantenic fucosylated structures with one or two sialic acid residues.

All of these post-translational modifications are extremely important for the biological activity of factor VII. In this regard, a recombinant protein is obtained by transforming mammalian cells (CHO, BHK-21 and others) with recombinant plasmids carrying the rhFVII gene. The resulting cell strain secretes rhFVII into the culture medium.

Blood coagulation factor VIIa promotes rapid blood coagulation, acting in the coagulation cascade at later stages compared to factor VIII. Therefore, rhFVIIa is mainly used to treat patients with hemophilia A having inhibitors (antibodies) to factor VIII, and patients with hemophilia B having inhibitors to factor IX. In more rare cases, rhFVIIa is also used when the level of FVIIa expression is insufficient to ensure normal coagulation rate. As a result, rhFVIIa is used to prevent hemorrhage during severe bleeding of various etiologies. In particular, it is used in obstetric and surgical practice (Gastroenterol. 2002; 123: 118-26); to stop internal bleeding after surgery (British Journal of Haematology 1999; 107: 677-678); in transplantation of hematopoietic stem cells (J Thromb Haemost. 2005 Sep; 3 (9): 1935-44); various injuries (Neurol Neurochir Pol. 2004 Nov-Dec; 38 (6): 527-31), after open heart surgery (Paediatr Anaesth. 2005 Mar; 15 (3): 235-40), etc.

Currently, several biotechnological systems for producing this protein are known. US Pat. No. 6,329,176 discloses a plasmid pLN174 containing a factor VII gene and a producer strain based thereon. The disadvantage of this system is the low level of protein expression achieved using the indicated plasmid (0.18 μg / ml). US Pat. No. 5,580,560 proposes the use of coexpression with the KEX-2 protease, which increases the protein yield by almost 5 times (0.90 μg / ml), but it is possible to obtain inactive forms of the protein that do not possess coagulation activity due to proteolytic degradation.

There are also a number of producers that make it possible to obtain factor VII, which differs from the natural substitution of one or more amino acids (see, for example, RF patents 2325401, 2338752, US patents 6960657, 6905683). However, when using such proteins to obtain drugs, there remains a risk of an immune response.

The plasmid containing the factor VII gene and the producer strain based on it, used to obtain this protein in mammalian cells, are also described in RF patent 2122583. The disadvantage of this plasmid is the complexity of assembling from a large number of elements of the DNA sequence encoding the desired protein, as well as a small yield of the target protein (about 1 μg / ml).

The closest analogue of the present invention is the plasmid pEFZeoF7 containing the factor VII gene and a producer based on it, used to obtain this protein, obtained by the method described in RF patent 2337965. The level of secretion of recombinant factor VII in this system was 40 μg / ml, which gave grounds for the possibility of its use in the industrial production of protein. However, tests for the stability of production and proliferation of the described cell line showed the impossibility of its use in the production process due to a sharp drop in both productivity and proliferative activity of the line for only a few passages. These properties indicate insufficient stability of the clone. Thus, the task of constructing plasmid DNA and creating a stable producer strain based on it, which allows one to obtain a protein with a good yield, remains relevant.

The present invention solves the problem of constructing plasmid DNA containing the sequence of recombinant factor VII, and creating a cell line (strain producer), which allows to obtain recombinant factor VII in good yield (at a constant level of 2-3 mg / l) and maintains its performance over a large the number of passages (at least 30), which is important and necessary for the industrial production of protein.

The problem is solved by constructing recombinant plasmid DNA pAR271, consisting of 12125 bp and the cell line Mesocricetus auratus BHK-21 k.13 (2H7), which allows to obtain rhFVII with a stably high yield, sufficient for industrial production.

The introduction of various genetic elements of the eukaryotic genome into recombinant constructs used for subsequent transfection of mammalian cells can have a positive effect on the stability of the transgene in the genome of the recipient cell, significantly improving the growth characteristics of the culture (SCMakrides Gene Transfer and Expression in Mammalian Cells, (2003) Elsevier Science, chapters 10-12). Similar elements, in particular MAR, are used to create industrial producer strains for which the critical parameter determining the possibility of its use in production is the stability of the level of production and the growth rate of the culture.

Elements of attachment to the nuclear matrix MAR (Matrix Attachment Regions) are fragments of eukaryotic DNA with a length of 300-3000 bp and play a key role in organizing nuclear and chromosomal architecture. At interphase, they fix chromatin with proteins that form the nuclear matrix, separating the eukaryotic genome into independent chromatin loops (Mirkovitch, J., Mirault, M.E., and Laemmli, U.K. (1984) Cell 39, 223-232.). Due to the colocalization of MAR elements with regions of active transcription and regulatory elements in the genome, the former positively affect the transgene transcription level by attracting transcription activators and controlling the availability of DNA in chromatin. In addition to these properties, MAR elements, such as the MAR lysozyme gene of birds, can prevent a gradual decrease in transgenic promoter activity, which significantly increases the stability of the transgene expression level (Allen, GC, Spiker, S., and Thompson, WF (2000) Plant Mol. Biol. 43, 361-376).

In the process of constructing the plasmid, we found that the use of MAR in a plasmid containing the hFVII gene allows maintaining the expression level of this protein with the greatest stability.

The advantages of the invention include, firstly, the use of a chemical-enzymatic method for the synthesis of the hFVII gene, which makes it possible to select the optimal codon composition of the gene and to avoid the presence of DNA structures in the sequence that impede the translation process, cryptic splicing sites, and polyadenylation. Optimization of the primary structure of the gene and the use of a combination of the promoter of the human translation elongation factor EF-1α and the CMV cytomegalovirus transcription enhancer, as well as the DNA fragment that secures attachment to the MAR nuclear matrix, can significantly increase the level of factor VII secretion into the culture medium, and, accordingly, the final yield target product.

Recombinant plasmid DNA pAP271 contains the following sequences: MAR region of attachment to the nuclear matrix of the bird lysozyme gene, enhancer of transcription of the CMV virus, promoter of the gene for translational elongation factor EF-1α, cDNA of the hFVII gene, internal translation initiation site IRES, DHFR gene, polyadenyl signal , a cassette containing all the necessary elements for the expression of the aminoglycoside-3'-phosphotransferase (ARH) gene, which ensures the resistance of the producer cell strain to geneticin, and cassettes for expression in ba cells the bacterium of the β-lactamase gene, which provides resistance to ampicillin. Plasmid pAP271 (figure 1) encoding a factor VII polypeptide is characterized by the following features:

- consists of 12125 bp,

- has a molecular weight of 7.49 MDa,

- encodes a polypeptide of human coagulability factor VII,

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

- contains unique recognition sites for the following restriction endonucleases: AgeI (850 bp), BbvCI (1657 bp), BmgBI (4202 bp), BssHII (6672 bp), Eco47III (11269 bp) bp), EcoRI (10929 bp), EcoRV (11863 bp), FseI (1455 bp), NotI (4812 bp), RsrII (6790 bp), SbfI (2330 bp), SfiI (6027 bp), Tth111I (6390 bp), XcmI (2404 bp).

The cell line Mesocricetus auratus BHK-21 k.13 (2H7) is a producer of recombinant human blood coagulation factor VII, which is a derivative of the fibroblasts of the kidneys of the newborn Syrian hamster Mesocricetus auratus VPK-21 k.13 characterized by the following features:

Morphological signs. Cells have typical morphological features of fibroblasts, are an adherent culture. This is a pseudodiploid line (2n = 44), the range of variation in the number of chromosomes is 35-45, the modal number of chromosomes is 40, the number of polyploids is not more than 14%.

Cultural signs. Cells grow in a monolayer on simple nutrient media, for example, on a minimal medium. Needle EMEM.

Physiological and biochemical characteristics. Cells grow at a temperature of 37 ° C, pH 6.7-7.3.

Antibiotic resistance. Cells are resistant to geneticin at a concentration of 800 μg / ml.

The strain of the cell line Mesocricetus auratus BHK-21 k.13 (2H7) was deposited at the Institute of Bioorganic Chemistry. Academicians M.M.Shemyakin and Yu.A. Ovchinnikov of the Russian Academy of Sciences March 22, 2008 (collection code E-002).

The claimed invention is illustrated in the following figures:

Figure 1 presents a map of recombinant plasmid DNA pAP271 used to create a producer strain of recombinant human blood coagulation factor VII.

Figure 2 presents:

A. SEQ ID N1 — amino acid sequence of human coagulation Factor VII, 444 a.k.

B. SEQ ID N2 - optimized gene sequence of coagulation factor VII of a person, 1335 bp

Figure 3 (a and b) presents the scheme for obtaining the vector construct pAP271 for expression of recombinant human factor VII coagulation.

The invention is illustrated by the following examples.

Example 1

Construction of recombinant plasmid DNA pAP271.

The nucleotide sequence corresponding to the cDNA of the human coagulation factor VII gene is obtained by chemical-enzymatic synthesis. To do this, the optimized gene sequence is divided into overlapping fragments of about 50 bp in size. The chemical synthesis of oligonucleotides corresponding to these fragments is carried out using the solid-phase phosphamidite method using the ASM-102U synthesizer (BIOSSET, Novosibirsk). The resulting oligonucleotides are subjected to 5'-terminal phosphorylation using T4 polynucleotide kinase (NEB, USA). Phosphorylated oligonucleotides are mixed in an equimolar ratio in 50 μl of a buffer containing 20 mM Tris-HCl, pH 7.56, 10 mM MgCl ₂ , 0.2 mM rATP, 10 mM dithiotreite, heated to 65 ° C, slowly cooled to 37 ° C for an hour and add T4 DNA ligase. The DNA ligation reaction is carried out for 12 hours at 37 ° C, 0.1 μl of the resulting solution is used as a matrix in the polymerase chain reaction (PCR) in the presence of thermostable Pfu DNA polymerase and specific primers:

F7_fwd 5'-TAG GCTAGC CGCCACCATGGTGAGCCAGGCACTGAG-3 'and

F7_rev 5'-CG ACGCGT TATTAGGGAAAGGGAGCTCTCAGCAG-3 '.

The DNA fragment synthesized in this way contains the cDNA sequence of the human coagulation factor VII gene flanked by the restriction enzyme recognition sites NheI and MluI — SEQ ID N2 (FIG. 2B). The amplification product is hydrolyzed with restriction enzymes NheI and MluI, purified by electrophoresis on a 1% agarose gel, a DNA strip of about 1350 bp. isolated from the gel by electroelution and precipitated DNA from the solution with ethanol.

To obtain vector DNA pAR271 used a series of consecutive stages of cloning (figure 3). Initially, the pIRES vector was used (Clontech, USA), into which the previously synthesized cDNA sequence of the factor VII gene was cloned at the restriction sites NheI and MluI. Further, using the XbaI and NotI restriction sites, the mouse dihydrofolate reductase gene was obtained by cloning the mouse by PCR using genomic DNA isolated from SP2 / 0 mouse myeloma cells and specific primers

DHFR_fwd 5'-CC TCTAGA TGGTTGTGGCCATATTATCATCGTGTTTTTCAAAG-3 'and

DHFR_rev 5'-GGAA GCGGCCGC TTAGTCTTTCTTCTCGTAGACTTCA-3 '

followed by site-directed mutagenesis to optimize the consensus sequence surrounded by the initiator codon of the IRES element using the QuikChange® II XL kit (Stratagene, USA) and the following oligonucleotides:

D3_fwd

5'-CCTTTGAAAAACACGATGATAATATGGCCACAACCATGGTTCGACCATTGAACTGCATC-3 'and

D3_rev

5'-GATGCAGTTCAATGGTCGAACCATGGTTGTGGCCATATTATCATCGTGTTTTTTCAAAGG-3 '.

The primary structure of the pAP232 intermediate vector construct in the region of the initiator codon of the IRES element was confirmed by Sanger sequencing.

Plasmid pAP232 was further sequentially treated with BglII restriction endonuclease and T4 DNA polymerase and the resulting vector was used in the ligation reaction with the PCR product obtained using MAR-specific primers

MAR_fwd 5'-GGGGATCCGTAATACAATTGTACCAGGTTTTGGTTTATTAC-3 'and

MAR_rev 5'-GAAAACAATATATTTCCAAATGAAAAAAAAATCTGATAAAAAG-3 '.

The selection of bacterial cells XL-10 Gold obtained after transformation of the ligase mixture was carried out in such a way as to reveal plasmid pAP257 DNA containing the MAR sequence in genomic orientation with respect to the promoter.

The PCR fragment of the DNA, which is a promoter of human translational elongation factor promoter obtained using primers, was cloned into pGL3Basic plasmid DNA (Promega, USA) at the BglII and HindIII sites

EF_fwd 5'-GAC AGATCT GGCTCCGGTGCCCGTCAGTGGGCAGAG-3 'and

EF_rev 5'-CA AAGCTT GCGACCACGTGTTCACGACACCTGAAATGG-3 '

and genomic DNA of human cells HEK293. The pAP262 vector thus obtained was linearized with a KpnI restriction endonuclease and the cytomegalovirus CMV transcription enhancer cloned by PCR using plasmid DNA pIRES and specific primers was cloned

CMV_fwd 5'-CGG GGTACC GCGTTACATAACTTACGGTAA-3 'and

CMV_rev 5'-CGG GGTACC AAAACAAACTCCCATTGACGTCAA-3 '.

About 1950 bp DNA fragment was cloned into the obtained pAP264 plasmid at the HindIII site, obtained after processing of the pAP257 vector DNA with HindIII restriction enzyme. The selection of recombinant plasmids was performed in such a way as to identify the vector pAR270 DNA containing the sequence of the gene of factor VII in direct relation to the promoter orientation. From the obtained pAP270 vector, a DNA fragment of about 2350 bp was subsequently isolated resulting from sequential treatment with NotI restriction enzyme, T4 DNA polymerase and limited hydrolysis of BglII restriction endonuclease under conditions of enzyme deficiency. Plasmid pAP257 treated with SpeI restriction enzyme, T4 DNA polymerase and BglII restriction endonuclease was used as vector DNA. These fragments were ligated with T4 DNA ligase; the resulting vector was named pAP271. Subsequently, the primary sequence of the obtained plasmid DNA was confirmed by the Sanger sequencing method. According to sequencing data, a plasmid was selected, the nucleotide and its corresponding amino acid sequence of the NheI - MluI fragment of which are completely identical to the cDNA sequence of the human coagulation factor VII gene, which was used in further work.

Example 2

Obtaining a cell line of Mesocricetus auratus BHK-21 k.13 (2H7), stably producing recombinant human coagulation factor VII.

To obtain a cell line stably producing recombinant human blood coagulability factor VII (rhFVII), cells of the Mesocricetus auratus BHK-21 line 13 transfect plasmid pAR271 plasmid DNA linearized at the BspHI restriction site, for example, by lipofection using jetPEI ™ reagent (Polyplus reagent (Polyplus Inc., USA).

Cells of Mesocricetus auratus BHK-21 k.13 are seeded on a Petri dish (for example, 10 cm in diameter) with a density of 2 × 10 ⁴ cells / cm ² in 12 ml of DMEM medium containing 10% bovine fetal serum and incubated for 24 hours at 37 ° C in a CO ₂ incubator. Transfection is then carried out using 15 μg of linearized at the BspHI restriction site plasmid pAP271 and 30 μl of jetPEI ™ reagent according to the manufacturer's recommendations. 48 hours after transfection, geneticin is selected on an antibiotic at a concentration of 800 μg / ml of clones of stably transfected cells. A change in the selective medium containing the antibiotic is carried out regularly every 3-4 days. Starting from the 7th day, the wells are analyzed for the presence of clones of actively dividing cells that are resistant to geneticin. Separately growing clones, having reached a size of 1-2 mm, are successively transplanted into 96-, then to 24- and 6-well plates as 80-90% confluency is achieved.

Clone productivity is assessed by enzyme-linked immunosorbent assay (ELISA) or immunoblot using monoclonal or polyclonal antibodies to human coagulation factor VII. For this, in wells in which the cells have reached 80-90% confluency, the medium is replaced with fresh medium containing an additional 10 μg / ml of vitamin K1 and incubated for 72 hours at 37 ° C in a CO ₂ incubator. The supernatant used in ELISA assay using, e.g., a set ^{Imubind ® FactorVII ELISA (American Diagnostica Inc.} , USA).

Wells with clones expressing rhFVII at a level of 1-2 mg / l / 24 hours are additionally cloned by limiting dilution. The most productive clones are used in the stability test for factor VII production. For this purpose, clones were cultured for 50-100 generations, periodically evaluating the level of rhFVII expression by ELISA.

Thus, one of the most productive and stable clones of Mesocricetus auratus BHK 21 k.13 (2H7) was obtained, which was then used to create a master bank and subsequent production of recombinant human blood coagulation factor VII. The protein expression level for this clone is 2-3 mg / L.

The preparation of recombinant activated human coagulability factor VII is based on the long-term cultivation of the Mesocricetus auratus BHK 21 cell line 13 k.13 (2H7), for example, for 30 days, with daily selection of culture fluid. The culture fluid is used to further isolate rhFVIIa using a series of successive stages: filtration, anion exchange chromatography in a gradient of salt concentration, affinity chromatography, anion exchange chromatography in a gradient of calcium concentration, during which factor VII is activated, gel filtration chromatography.

The specific activity of rhFVIIa thus isolated, measured by the standard coagulometric method, is 44000-64000 IU / mg.

Thus, the present invention allows to obtain a recombinant activated coagulation factor of human blood VIIa with properties identical to those of a natural polypeptide. The resulting rhFVIIa can then be used for the manufacture of drugs.

Claims (2)

1. Recombinant plasmid DNA pAP271 encoding a polypeptide with a sequence of human coagulation factor VII, having a size of 12125 bp, with the physical map shown in figure 1, and consisting of the following elements:
an optimized sequence encoding a human blood coagulation factor VII polypeptide,
MAR - areas of attachment to the nuclear matrix of the lysozyme gene of birds,
CMV virus transcription enhancer,
promoter of the gene for translational elongation factor EF-1α person,
encephalomyocarditis virus internal translation initiation site,
mouse DHFR gene,
SV40 polyadenylation signal,
a cassette containing all the necessary elements for the expression of the aminoglycoside 3'-phosphotransferase (ARN) gene, which ensures the resistance of the producer cell strain to geneticin,
cassettes for the expression in the bacterial cells of the β-lactamase gene, which provides resistance to ampicillin,
unique recognition sites for the following restriction endonucleases: AgeI (850 bp), BbvCI (1657 bp), BmgBI (4202 bp), BssHII (6672 bp), Eco47III (11269 bp ), EcoRI (10929 bp), EcoRV (11863 bp), FseI (1455 bp), NotI (4812 bp), RsrII (6790 bp), SbfI (2330 bp), SfiI (6027 bp), Tth111I (6390 bp), XcmI (2404 bp). 2. The cell line Mesocricetus auratus BHK 21 k.13 (2H7) is a producer of recombinant human blood coagulation factor VII obtained by transforming the cell line Mesocricetus auratus BHK 21 k.13 recombinant plasmid DNA pAP271 according to claim 1.

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