RU2749459C1 - UNIVERSAL INTEGRATION VECTOR pVEAL AND RECOMBINANT PLASMID pVEAL-15742, PROVIDING SYNTHESIS AND SECRETION OF scFv-Fc ANTIBODIES AGAINST EBOLA VIRUS ADI-15742 IN MAMMALIAN CELLS AND OBTAINED USING pVEAL VECTOR - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to biotechnology. Described is the universal integration vector pVEAL and the recombinant plasmid pVEAL-15742, which provides the synthesis and secretion of scFv-Fc antibodies ADI-15742 capable of neutralizing the Ebola virus in mammalian cells. The universal integration vector pVEAL is used to create target plasmid constructs that provide for the synthesis and secretion of scFv-Fc monoclonal antibodies, has the nucleotide sequence SEQ ID No. 1 and contains elements in accordance with the physical and genetic map shown in Fig. 1. The plasmid genetic construct pVEAL-15742, which provides the synthesis and secretion of the target protein scFv-Fc of the anti-Ebola virus ADI 15742 antibody, has the nucleotide sequence of SEQ ID No. 2 and contains elements in accordance with the physical and genetic map shown in Fig. 2.EFFECT: creation of a universal integration vector pVEAL and a plasmid genetic construct pVEAL-15742, which provides the synthesis and secretion of the target protein scFv-Fc of the ADI 15742 antibody against the Ebola virus in mammalian CHO-K1 cells, obtained using the specified universal vector pVEAL.2 cl, 6 dwg, 5 ex

Description

The invention relates to a universal integration vector pVEAL and a recombinant plasmid pVEAL-15742, which provides the synthesis and secretion of scFv-Fc antibodies ADI-15742 capable of neutralizing the Ebola virus in mammalian cells using the universal vector pVEAL and can be used in molecular genetics and biotechnology.

State of the art. Preparations based on monoclonal antibodies are the most promising tool for emergency counteraction to viruses due to their high specificity and minimal side effects. To date, at least 570 antibodies worldwide have undergone clinical trials (Lu R.M. et al. Development of therapeutic antibodies for the treatment of diseases. Journal of biomedical science. 2020; 27 (1): 1-30; Wong G. et al. Post-exposure therapy of filovirus infections. Trends Microbiol. 2014; 22 (8): 456-63; Saphire EO An update on the use of antibodies against the filoviruses. Immunotherapy. 2013; 5 (11): 1221 - 33). Significant technological advances have made the discovery and development of monoclonal antibody therapies faster and more effective.

et al. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC biotechnology. 2013, 13(1): 52; Girgis M.D. et al. Targeting CEA in pancreas cancer xenografts with a mutated scFv-Fc antibody fragment. EJNMMI research. 2011, 1(1): 1-10).Currently, the production of scFv-Fc antibodies is widespread. These are single-chain antibodies composed of variable fragments of the light and heavy chains of an antibody and a constant chain. The scFv-Fc format has several advantages over large fragments or whole antibodies: fewer cleavage sites for circulating proteolytic enzymes and, therefore, higher stability; The scFv-Fc variant involves the synthesis of a single chain, which facilitates the creation of vectors and constructs, simplifies the scheme of obtaining and testing, single-chain antibodies reach the target faster, and are cleared from the body faster (Huston JS et al. Protein engineering of single-chain Fv analogs and fusion proteins. Methods in enzymology 1991,203: 46-88; WO 1993/16185; US 5571894). Possible allergic reactions to the non-canonical structure are insignificant in the case of antibodies against infectious diseases. Such antibodies, first of all, should be effective

et al. High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. BMC biotechnology. 2013, 13 (1): 52; Girgis MD et al. Targeting CEA in pancreas cancer xenografts with a mutated scFv-Fc antibody fragment. EJNMMI research. 2011, 1 (1): 1-10).

One way to obtain monoclonal scFv-Fc antibodies is to insert nucleotide sequences encoding the variable sequences of the heavy and light chains of antibodies into integration vectors. These vectors allow the replication and expression of recombinant gene constructs in a suitable host by integration into the chromosome of the host. The integration vector includes: recombinant immunoglobulin domains, the origin of replication or autonomously replicating sequence, dominant marker sequences, signal sequences, sequences that control expression and transcription of recombinant genes, a system responsible for integration into the genome (RU 2469093 C2, RU 2639519). Transfection of mammalian cell lines with these vectors and their further cultivation allows cells to synthesize antibodies directly into the culture fluid (RU 2292353 C2).

Viral delivery systems and non-viral DNA vectors can act as an integration vector. Virus-based delivery systems have the following disadvantages: 1) virus preparations have a risk of being infected by infectious agents, including replication-competent virus; 2) the viral vector can cause undesirable cellular consequences, for example, acute immune, inflammatory or neurotoxic responses; 3) efficient genetic propagation of viral vectors can limit the size of the therapeutic load and may require genetic motives to regulate vector replication. In contrast, plasmid-based non-viral DNA vector preparations are relatively inexpensive to purify, largely non-immunogenic, and do not have severe restrictions on the sequences that can be delivered. The main problems of non-viral systems based on plasmids are as follows: 1) low rates of delivery of vectors to the nuclei of target cells; 2) low rates of integration of transgenes, usually with concomitant plasmid sequences that tend to suppress expression; 3) integration of multiple copies of the transgene, which can also suppress their expression (Hackett RV, et al. A transposon and transposase system for human application. Molecular therapy. 2010, 18 (4): 674-683).

The above main problems associated with non-viral vectors can be overcome using the SB (Sleeping Beauty) transposase-based system. This system is simple and does not require the creation of viral constructs. To ensure the integration of the sequence into the genome, two vectors are required, one must contain the sequence of the target insertion flanked by the IR / DR (L / R) sequences, the second vector must provide the synthesis of SB transposase. By increasing the enzyme activity, transposases SB10, SB11, SB100, etc. are isolated. (Hackett R. V., et al. A transposon and transposase system for human application. Molecular therapy. 2010, 18 (4): 674-683; Tipanee J. et al. Transposons: moving forward from preclinical studies to clinical trials. Human Gene Therapy. 2017,28 (11): 1087-1104). Advantages of transposase-based systems: biosafety, ease of use, the length of the inserted fragment is not limited, stable expression of the inserted gene.

The closest analogues. In the work (Oksanich A.S. et al. Construction of a plasmid vector for obtaining chimeric antibodies of a given specificity in mammalian cells. Zh.Microbiol. 2017, 6: 56-63), a universal plasmid vector is presented that makes it possible to obtain full-length chimeric antibodies of various specificities of various classes and isotypes. This vector is based on the well-known eukaryotic plasmid vector pCI-neo and includes a strong CMV promoter, antibiotic resistance genes, light and heavy chains of a given antibody. The vector is simple to construct; available and inexpensive methods are used to obtain ready-made antibody preparations. However, the final antibody yield is low (4 mg / L).

International application WO 2007/081336 describes a vector for the production of highly secreted recombinant proteins. The advantage of this invention is the triple leader sequence, the CMV promoter in combination with the regulatory element elongation factor-1, which should provide a significant level of protein synthesis. The vector also contains PolyA, SV40 ori, SV40 PA, pUC Origin Site, antibiotic resistance genes ampicillin, neomycin. In the description of the invention, there is no data on what kind of protein yield can be obtained.

The disadvantages of the above inventions are the lack of cis-regulatory elements. When cells are transfected with such vectors, the latter are randomly inserted into the chromosome; the conformation of the insertion site significantly affects gene transcription and its expression. Cis regulatory elements allow an increase in gene expression without affecting the chromatin structure in the insertion region. Cis-regulatory elements include: locus control region (LCR), scaffold / matrix attachment region (S / MAR), insulator, ubiquitous chromatin open element (UCOE), anti-repressor (STAR element).

The closest analogue (prototype) is a universal vector described in the Chinese patent (CN 105255895, IPC C12N 15/85, publ. 20.01.2016). The authors argue that the proposed expression system provides a high yield of the protein, the expression is stable and long-term. The vector is based on SB transposase, which makes it easy to integrate the desired sequence into the cell genome. The cis-regulatory element is the matrix attachment region - MAR2. The heavy and light chains of the antibody are linked by IRES. An "internal ribosome entry site" or "IRES" describes a sequence that functionally initiates translation and allows two cistrons (open reading frames) to be translated from a single transcript in an animal cell. The use of IRES elements in vector constructs has been previously described (Ramesh, N., et al. Nucl. Acids Res. 1996, 24: 2697-2700; Mosser, D.D. et al. BioTechniques. 1997, 22: 150-152). The CMV cytomegalovirus promoter was used to initiate transcription.

However, the known universal prototype vector provides the synthesis of only full-length antibodies, which, as mentioned earlier, are inferior in many parameters to antibodies of the scFv-Fc format.

The prototype of the recombinant plasmid that provides the synthesis of antibodies against the Ebola virus is the construct described in US patent (US 10640550, IPC A61P 31/14, publ. 05.05.2020). The vector constructed in this work contains a promoter with an enhancer, IRES, PolyA, antibiotic resistance genes ampicillin, neomycin. Integration of genetic material into the genome is possible using a transposase-based system. The production of antibodies can be carried out using hybridoma technology, or by transfection of eukaryotic cells with subsequent release of the synthesized antibodies into the culture fluid.

The disadvantages of this design are the absence of cis-regulatory elements, the need to insert full-length light and heavy antibody chains. The description of the vector does not indicate which promoter was used to initiate transcription, there is no data on the yield of the target protein, which does not allow judging the effectiveness of this construct.

The technical result of the claimed invention is the creation of a universal integration vector pVEAL and a plasmid genetic construct pVEAL-15742, providing the synthesis and secretion of the target protein - scFv-Fc antibody ADI 15742 against the Ebola virus (Anna Z. Wec, et al. Antibodies from a Human Survivor Define Sites of Vulnerability for Broad Protection against Ebolaviruses. Cell. 2017, 169 (5): 878-890) in mammalian CHO-K1 cells, obtained using the specified universal vector pVEAL, which is devoid of the disadvantages of the prototype.

The specified technical result is achieved in that the universal integration vector pVEAL serves to create target plasmid constructs that provide the synthesis and secretion of scFv-Fc monoclonal antibodies, has the nucleotide sequence SEQ ID No. 1 and contains, in accordance with the physical and genetic map shown in FIG. 1, the following items:

- site of the beginning of replication ori (8616-9204);

- IR / DR (L / R) sequences, which are part of the SB100 transposase-based system (139-403, 7832-8127);

- UCOE sequences preventing chromosomal silencing of the recombinant expression cassette (416-1549, 6272-7820);

- CMV Enhancer (2108-2487), CMV promoter (2488-2699) - the strongest most commonly used promoter in gene therapy constructs;

- Chimeric intron (2860-2992), which increases the efficiency of the CMV promoter;

- the leader peptide VI9, which ensures the export of the protein from the cell (3114-3164);

- a place for the insertion of variable fragments of the heavy and light chains of the antibody, connected by a peptide linker, at the sites of hydrolysis AfeI (3157), BamHI (3339);

- constant chain of a human IgG1 antibody (CH1-CH2-CH3) (3348-4031) with mutations M252Y, S254T, T256E, E333A, H433K / N434F. Mutations increase the half-life of an antibody by interacting with neonatal receptors;

- site of internal landing of the ribosome EMCV IRES (4046-4620);

- the Bleo sequence encoding the antibiotic resistance factor bleomycin (4625-4995);

- SV40 poly (A) signal sequence required for stabilization of mRNA transcripts (5042-5163);

- SV40 promoter (5921-6262), providing autonomous replication in mammalian cells, the combination with the CMV promoter significantly increases the yield of the protein;

- antibiotic resistance gene ampicillin AmpR and bacterial promoter of ampicillin resistance gene, allowing preparative plasmid production in E. coli (9375-10235).

The specified technical result is also achieved in that the plasmid genetic construct pVEAL-15742, providing the synthesis and secretion of the target protein - scFv-Fc antibodies ADI 15742 against the Ebola virus, has a nucleotide sequence of SEQ ID No. 2 and contains, in accordance with the physical and genetic map shown in FIG. 2, the following items:

- site of the beginning of replication ori (8616-9204);

- sequences IR / DR (L / R), which are part of the system based on transposase SB 100 (139-403, 8393-8688);

- UCOE sequences preventing chromosomal silencing of the recombinant expression cassette (416-1549, 6833-8381);

- CMV Enhancer (2108-2487), CMV promoter (2488-2699) - the strongest most commonly used promoter in gene therapy constructs;

- Chimeric intron (2860-2992), which increases the efficiency of the CMV promoter;

- the leader peptide VI9, which ensures the export of the protein from the cell (3114-3164);

- ADI-15742-VH, the sequence encoding the variable fragment of the heavy chain of the antibody 15742 (3165-3533);

- ADI-15742-VK, the sequence encoding the variable fragment of the light chain of the antibody 15742 (3579-3903);

- constant chain of a human IgG1 antibody (CH1-CH2-CH3) (3909-4592) with mutations M252Y, S254T, T256E, EZZZA, H433K / N434F. Mutations increase the half-life of an antibody by interacting with neonatal receptors;

- site of internal landing of the ribosome EMCV IRES (4607-5181);

- the Bleo sequence encoding the antibiotic resistance factor bleomycin (5186-5556);

- the SV40 poly (A) signal sequence required for the stabilization of mRNA transcripts (5603-5724);

- SV40 promoter (6482-6823), providing autonomous replication in mammalian cells, the combination with the CMV promoter significantly increases the yield of the protein;

- the gene for resistance to the antibiotic ampicillin AmpR and the bacterial promoter of the gene for resistance to ampicillin, allowing for preparative production of the plasmid in E. coli (9936-10796).

The claimed plasmids are significantly improved in comparison with the prototypes because transposase SB 100, which has a high enzyme activity, is used as an integration system (Mates L et al. Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009, 41 (6): 753-61) , there are strong cis-regulatory elements UCOE, preventing chromosomal silencing (Neville JJ et al. Ubiquitous Chromatin-opening Elements (UCOEs): Applications in biomanufacturing and gene therapy. Biotechnol Adv. 2017, 35 (5): 557-564), combination CMV promoter with enhancer, intron A and SV40 promoter significantly increases the protein yield (RU 2639519), a strong V19 leader peptide that increases the secretion of the recombinant protein (WO 2008/148519 A2), EMCV IRES-linked co-expression of the selection marker Bleo and antibody sequences can improve breeding process (RU 2639519), scFv-Fc format of antibodies facilitates the creation of a construct, simplifies the scheme for obtaining and testing the required antibody.

The invention is illustrated by the following graphical figures. FIG. 1 shows the physical and genetic map of the universal integration vector pVEAL. FIG. 2 shows the physical and genetic map of the integration vector pVEAL with the insert of the scFv-Fc antibody against the Ebola virus ADI-15742 - pVEAL-15742. FIG. 3 shows the determination of the minimum concentration of bleomycin required for the death of an untransfected host cell line. FIG. 4 shows an analysis of the neutralizing activity of the monoclonal scFv-Fc antibody ADI 15742 against Ebola virus. FIG. 5 shows an analysis of the culture medium of the scFv-Fc antibody 15742 producing strain. FIG. 6 shows the analysis of the scFv-Fc preparation of antibody 15742 after chromatography on protein A.

For a better understanding of the essence of the invention, below are examples (1-5) of its implementation. All standard genetic engineering and microbiological manipulations, as well as amplification and sequencing of DNA were carried out according to known methods (Maniatis T., Fritsch E, Sambrook J. Molecular cloning, M .: Mir, 1984; DNA cloning. Methods. Ed. By D. Glover, Translated from English, Moscow, Mir, 1988; Saiki RK et al. Science. 1988, 239 (4839): 487-491; Sanger F. et al. Proc. Nat. Acad. Sci. 1977, 74: 5463-5467).

Example 1. Construction of a universal integration vector pVEAL

The pT2HB acceptor vector (Addgene plasmid # 26557) was used to design the pVEAL-vector. The integration vector was obtained in several stages. At the first stage, the BamHI hydrolysis site was removed from the pT2HB vector using site-directed mutagenesis. For this, PCR was performed using primers:

и-

and

-

PCR of the PCR-pT2HB fragment was performed using a BIS PCR amplifier (BIS-N, Russia). The reaction mixture with a volume of 50 μl contained 2 μg of DNA (plasmids pT2HB), 10 pM of each primer (pT2HB-F, pT2HB-R), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP 2.5 mM each) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C 30 s - 58 ° C, 45 s - 72 ° C (25 cycles); 120 s - 72 ° C.

The reaction mixture containing the PCR-pT2HB fragment was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the band corresponding to the amplification product - 709 bp, visualized with ultraviolet radiation (UV), was excised from the agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the isolated PCR-pT2HB PCR product and the original pT2HB plasmid were treated with restriction endonucleases Psp124BI and KpnI according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 688 and 2859 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the method of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable with genotype F 'proA + B + lacIq Δ (lacZ) M15 zzf :: Tn10 (TetR) Δ (ara-leu) 7697 araD139 fhuA ΔlacX74 galK16 galElac El4Δ- Ф80d recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ (mrr-hsdRMS-mcrBC) supplied by Invitrogen (USA). For this, 10 μl of ligase mixture was added to 100 μl of cells, incubated on ice for 40 minutes. Thereafter, the cells were subjected to "temperature shock" at 42 ° C for 40 seconds. The cells were cooled on ice for 4 minutes, then 200 μl of SOC medium (20 g / l tryptone, 5 g / l yeast extract, 0.5 g / l NaCl, 250 mM KCl, 10 mM MgCl2) was added and incubated at 30 ° C for 60 minutes. At the end of incubation, the transformed cells were plated on a Petri dish with a solid LB nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pT2HB2 was obtained.

The next step was to insert the UCOE-R and UCOE-L sequences. Human genomic DNA was used to generate the UCOE-R and UCOE-L sequences. To amplify the UCOE-R sequence, PCR was performed using primers:

и-

and

-

PCR of the UCOE-R fragment was performed using a BIS PCR amplifier (BIS-N, Russia). The reaction mixture with a volume of 50 μl contained 2 μg of DNA (human genomic DNA), 10 pM of each primer (UCOE-RF, UCOE-RR), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP at 2.5 mM) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s - 59 ° C, 95 s - 72 ° C (30 cycles); 120 s - 72 ° C.

The reaction mixture containing the UCOE-R fragment was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the band corresponding to the amplification product - 1572 bp, visualized using ultraviolet radiation (UV), was excised from the agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then the isolated PCR product UCOE-R was treated with restriction endonucleases BseX3I and BamHI, plasmid pT2HB2 was treated with restriction endonucleases BseX3I and BglII according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 1555 and 3529 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the method of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pT2HB2-UCOE-R was obtained.

Amplification of the UCOE-L sequence was carried out by PCR using primers:

и-

and

-

PCR of the UCOE-L fragment was performed using a BIS PCR amplifier (BIS-N, Russia). The reaction mixture with a volume of 50 μl contained 2 μg of DNA (human genomic DNA), 10 pM of each primer (UCOE-LF, UCOE-LR), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP at 2.5 mM) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s - 59 ° C, 97 s - 72 ° C (30 cycles); 120 s - 72 ° C.

The reaction mixture containing the UCOE-L fragment was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the band corresponding to the amplification product - 1593 bp, visualized using ultraviolet radiation (UV), was excised from the agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the isolated PCR product UCOE-L and plasmid pT2HB2-UCOE-R were treated with restriction endonucleases HindIII and BseX3I according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 1581 and 5051 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the procedure of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pT2HB2-UCOE-RL was obtained.

Further construction of the vector consisted in the insertion of the V19-ADI sequence (synthesized by order of DNA-Synthesis LLC), which encodes the V19 leader peptide and the unique AfeI and BamHI hydrolysis sites, through which the chains of the required antibody and scFV-Fc fragment will be inserted.

Ligation of the V19-ADI sequence to a commercial pJet vector (CloneJET PCR Cloning Kit, Thermo Scientific) was performed according to the manufacturer's instructions. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The pJet-V19-ADI construct was obtained.

Next, the V19-ADI sequence was inserted into the pCI-neo-hEST2 vector (# 1781). This vector was chosen because it contains a strong eukaryotic CMV promoter, as well as a set of antibiotic resistance genes that make it convenient to work with.

Vector pCI-neo-hEST2 and pJet-V19-ADI were treated with restriction endonucleases PspCI and SalI according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% and 2% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 5467 and 182 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the method of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pCI-neo-hEST2-V19-ADI was obtained.

Next, the cloning of the sequence encoding the constant part of the heavy chain of the human antibody (CH) Human IgG1 into the vector pCI-neo-hEST2-V19-ADI was carried out. Primers were used to amplify the sequence encoding the constant part of the heavy chain of a human antibody:

и-

and

-

The plasmid pMAB-CH-100VH was used as a DNA template.

PCR of a PCR-Human IgG1 fragment was performed using a BIS PCR amplifier manufactured by OOO BIS-N (Russia). The reaction mixture with a volume of 50 μl contained 2 μg of DNA (rMAB-CH-100VH), 10 pM of each primer (Hinge_Bspl3I_F, CH3_SalI_R), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dTCP 2.5 mM) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s - 60 ° C, 43 s - 72 ° C (30 cycles); 120 s - 72 ° C.

The reaction mixture containing the PCR-Human IgG1 fragment was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the band corresponding to the amplification product - 708 bp, visualized with ultraviolet radiation (UV), was excised from the agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the isolated PCR product PCR-Human IgG1 and plasmid pCI-neo-hEST2-V19-ADI were treated with restriction endonucleases Bsp13I and SalI according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 690 and 5635 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the procedure of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pCI-neo-hEST2-V19-ADI-CH-21 was obtained.

Next, the gene for resistance to the antibiotic bleomycin and the EMCV IRES sequence were inserted. Primers used for amplification:

-

-

PCR of the PCR-Bleo-scFv fragment was performed using a BIS PCR amplifier (BIS-N, Russia). The reaction mixture with a volume of 50 μl contained 2 μg of DNA (FUGW, Addgene plasmid # 14883), 10 pM of each primer (Bleo-scFv-F, Bleo-scFv-R), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP 2.5 mM each) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s - 58 ° C, 25 s - 72 ° C (30 cycles); 120 s - 72 ° C.

The reaction mixture containing the PCR-Bleo-scFv fragment was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, a band corresponding to the amplification product - 400 bp, visualized with ultraviolet (UV), was excised from an agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the isolated PCR product PCR-Bleo-scFv and plasmid pJet-EMCV-IRES-V17-100VH-CH-2018 were treated with restriction endonucleases Msp20I and AsuNHI according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the bands corresponding to the required fragments - 380 and 4578 bp, visualized with ultraviolet (UV), were excised from the agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the method of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pJet-IRES-Bleo was obtained.

PCR of the EMCV-IRES-BleoR fragment was performed using a BIS PCR amplifier (BIS-N, Russia). Primers used for amplification:

и-

and

-

The reaction mixture with a volume of 50 μl contained 2 μg of DNA (pJet-IRES-Bleo), 10 pM of each primer (IRES-scFv-R and IRES-Bleo-scFv-R), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), mixture deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP 2.5 mM each) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s -58 ° C, 60 s - 72 ° C (30 cycles); 120 s - 72 ° C.

The reaction mixture containing the EMCV-IRES-BleoR fragment was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the band corresponding to the amplification product - 984 bp, visualized by ultraviolet (UV), was excised from the agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the isolated PCR product EMCV-IRES-BleoR and plasmid pCI-neo-hEST2-V19-ADI-CH-21 were treated with restriction endonucleases XhoI and NotI according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 965 and 6300 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the method of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pCI-neo-hEST2-V19-ADI-CH-IRES-BLEO-21 was obtained.

The last step in obtaining the target vector pVEAL was the combination of the pT2HB2-UCOE-RL and pCI-neo-hEST2-V19-ADI-CH-IRES-BLEO-21 constructs. Plasmid pT2HB2-UCOE-RL was treated with restriction endonucleases BamHI and Mox20I, plasmid pCI-neo-scFv-ADI-CH-IRES-BLEO-21 was treated with restriction endonucleases BglII and HpaI.

Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 6617 and 4297 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the procedure of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was carried out according to the Sanger method at the Genomics Center for Collective Use of the Siberian Branch of the Russian Academy of Sciences (Novosibirsk).

The insertion of the sequences of the variable fragments of the heavy and light chains of any antibody into the universal vector pVEAl is carried out at the unique AfeI and BamHI hydrolysis sites.

Example 2. Obtaining the genetic construct pVEAL-15742 encoding the scFv-Fc antibody ADI-15742 for transfection of the CHO-K1 cell line.

To confirm the effectiveness of the universal integration vector pVEAL, the variable sequences of the heavy and light chains of the ADI-15742 antibody and the sc-Fv fragment that bind them were inserted.

To begin with, the heavy and light chains of the ADI-15742 antibody were amplified. PCR of the PCR-ADI-15742VH fragment was carried out from the synthesized sequence ADI-15742VH (DNA-synthesis LLC) using primers:

и-

and

-

PCR fragment of PCR-ADI-15742VK with synthesized sequence ADI-15742VK (DNA-synthesis LLC) using primers:

и-

and

-

PCR of fragments PCR-ADI-15742VH, PCR-ADI-15742VK was carried out using a PCR amplifier "BIS" manufactured by OOO BIS-N (Russia). Reaction mixtures with a volume of 50 μl contained 2 μg of DNA (ADI-15742VH / ADI-15742VK), 10 pM of each primer (V19-ADI-F, V19-ADI-LR / V19-ADI-LF, V19-ADI-R), 5 μl 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP 2.5 mM each) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s -58 ° C, 30 s - 72 ° C (30 cycles); 120 s - 72 ° C.

Reaction mixtures containing fragments of PCR-ADI-15742VH, PCR-ADI-15742VK were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the amplification products - 487 and 384 bp, visualized with ultraviolet (UV), were excised from an agarose gel and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, annealing and subsequent amplification of the sequences PCR-ADI-15742VH, PCR-ADI-15742VK were carried out using primers V19-ADI-F and V19-ADI-R using a PCR amplifier "BIS" manufactured by OOO BIS-N (Russia). The reaction mixture with a volume of 50 μl contained 2 μg of DNA for each fragment (PCR-ADI-15742VH, PCR-ADI-15742VK), 10 pM of each primer (V19-ADI-F and V19-ADI-R), 5 μl of 10xTaq buffer (Sibenzyme (Russia)), a mixture of deoxynucleotide triphosphates (dATP, dCTP, dTTP, dGTP 2.5 mM each) and 1 unit. DNA polymerase Taq-SE from Sibenzyme (Russia); the reaction was carried out with the following parameters: 3 min - 95 ° C; 30 s - 95 ° C, 30 s -58 ° C, 51 s - 72 ° C (30 cycles); 120 s - 72 ° C.

The reaction mixture containing the amplified fragment scFv-ADI-15742 was separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, the band corresponding to the amplification product - 851 bp, visualized by ultraviolet (UV), was excised from the agarose gel, and placed in a 1.5 ml tube. The fragment was isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

The universal integration vector pVEAL and the scFv-ADI-15742 fragment were treated with restriction endonucleases AfeI and BamHI according to the manufacturer's protocol (Sibenzyme (Russia)). Restricted fragments were separated by electrophoresis in 1% agarose gel with the addition of ethidium bromide (0.4 μg / ml). To isolate DNA, bands corresponding to the required fragments - 10732 and 743 bp, visualized with ultraviolet (UV), were excised from an agarose gel, and placed in 1.5 ml tubes. Fragments were isolated from the gel using the Qiagen Gel Extraction Kit (Germany) according to the manufacturer's instructions.

Then, the fragments were ligated with T4 phage DNA ligase (Sibenzyme (Russia)) in a standard buffer solution according to the method of the enzyme manufacturer. The resulting ligase mixture was used to transform "competent" cells of E. coli strain NEB Stable as described above. The transformed cells were plated on a Petri dish with LB solid nutrient medium (LB medium with 1.5% agar) containing an antibiotic (ampicillin, 50-100 μg / ml). The Petri dish was placed in a thermostat for 18 hours at 30 ° C. Individual clones of transformants were inoculated into 5 ml of LB nutrient broth supplemented with ampicillin at a concentration of 50 μg / ml, grown for 18 hours at 30 ° C, and plasmid DNA was isolated using Plasmid Mini Kit, Qiagen (Germany) in accordance with manufacturer's instructions. The primary structure of the resulting plasmids was confirmed by sequencing. Sequencing was performed according to the Sanger method at the Genomics Center for Collective Use, Siberian Branch of the Russian Academy of Sciences (Novosibirsk). As a result, the plasmid pVEAL-15742 was obtained.

Example 3. Obtaining clones-producers of scFv-Fc antibody 15742

To obtain producer cells, CHO-K1 cells (clone K1 of the CHO line isolated from Chinese hamster ovary) were selected. The use of these cells is based on the experience of their commercial use to create producers of therapeutic proteins, including antibodies. The line was maintained in a medium based on DMEM / F12 supplemented with 10% heat-inactivated (30 min at 56 ° C) fetal bovine blood serum (FBS), L-glutamine. Upon reaching the monolayer, the cells were transplanted.

To obtain a stable cell line that synthesizes the recombinant protein, the minimum concentration of the selective antibiotic bleomycin required for the death of the untransfected host cell line was determined. To determine the IC50 and IC100 of bleomycin, the cells were seeded into a 12-well culture plate with DMEM / F12 medium supplemented with 10% fetal bovine blood serum (FBS) and L-glutamine, after reaching 90% confluence of the monolayer, the culture medium was replaced with 2% DMEM / F12 with the addition of the antibiotic bleomycin (50-700 μg / ml). Within 14 days, every 48-72 hours, the culture medium in the wells was replaced with 2% DMEM / F12 with the addition of bleomycin (50-700 μg / ml).

When bleomycin was added, cells sensitive to it showed the following morphological changes:

• Increase in size (similar to the effects of cytomegalovirus infecting cells)

• Abnormal cell shape, including the appearance of long appendages

• The presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and Golgi apparatus)

• Destruction of plasma and nuclear membrane (the appearance of many holes in these membranes). Eventually, the cells will completely collapse, and only cellular debris will remain.

These changes are illustrated in FIG. 3. In FIG. 3a shows a photograph of cells not exposed to bleomycin - positive control K +. FIG. Figures 3b - 3i show photographs of cells exposed to bleomycin in an amount of 50 to 700 μg / ml. These figures show that with an increase in the concentration of the antibiotic, the cells change significantly morphologically. FIG. 3d, at a bleomycin concentration of 300 μg / ml, it can be seen that about half of the cells retain their viability and their inherent morphology. FIG. 3f, 3g cells almost completely died, altered and destroyed cells predominate.

Thus, the semi-inhibitory and inhibitory doses of bleomycin for CHO-K1 were established (IC50 = 325 μg / ml, IC100 = 475 μg / ml).

To insert the expression cassette, CHO-K1 cells were co-transfected by cation-lipid transfection (Lipofectamine 3000, Invitrogen) with two plasmids. The first contained the sequence of the scFv-Fc antibody 15742, pVEAL-15742, the second contained the sequence of transposase SB100, pCMV (CAT) T7-SB100. After transfection, the cells were incubated for 96 hours in a CO2 incubator according to the manufacturer's recommendations Lipofectamine 3000. Then, the medium was replaced with a supporting medium (containing 2% fetal bovine serum) with the addition of a selective antibiotic bleomycin (475 μg / ml). This manipulation was repeated every 72-96 hours for 2-3 weeks until the cells died in the negative control. Then, one passage was made on culture dishes and plated in 96-well plates, one cell per well.

4 clones 15742 were obtained with a neutralizing titer determined for IC90 (culture medium analyzed) from 1: 8 to 1: 32768. The virus neutralization reaction was carried out on a pseudoviral system.

Example 4. Assay of scFv-Fc neutralizing activity of anti-Ebola monoclonal antibody ADI15742

Antibody 15742 preparations were titrated with a step of 4, incubated for 30 min in a CO2 incubator with a mixture of pseudoviral particles of the Ebola virus (50 μl, physical titer 5 × 10 ⁸ particles) and transferred to a 96-well flat-bottom plate to a HEK293T cell culture suspension (50 μl, 100 thousand / ml). Incubated for 48 h in a CO2 incubator. After the time elapsed, the growth medium was removed from the wells of the plate, washed with 100 μl PBS, added 40 μl 1 × lysis buffer (Promega, USA), incubated for 10 minutes at room temperature, suspended and transferred 35 μl into an optical plate, 35 μl LAR ( Promega, USA), and the results were read using a luminometer (LuMate). The analysis results for one of the clones are shown in FIG. 4. The graph shown in FIG. 4 depicts the dependence of the neutralizing activity of the scFv-Fc antibody against Ebola virus ADI 15742, determined for IC90, on the degree of drug dilution. At a dilution of 1: 8, the antibody preparation has one hundred percent neutralizing activity. At a dilution of up to 1: 32768, the neutralizing activity is 75%.

Example 5. Production and purification of scFv-Fc monoclonal antibody ADI15742

Each individual clone-producer of the recombinant scFv-Fc antibody 15742 was produced in DMEM / F12 culture medium with 10% fetal bovine serum. Cultivation of producer clones was carried out in a volume of 200 ml in roller bottles at 37 ° C for 10 days. Then the medium was replaced with a maintenance medium containing 2% fetal calf serum. The cultivation was continued as long as the cells remained viable. Then the resulting culture fluid was discarded, and the cell debris was precipitated by centrifugation at 6000 g, 4 ° C for 15 min.

The recombinant antibodies were purified by affinity chromatography using a sorbent containing protein A on its surface (MabSelect SuRe, GE Healthcare, USA) and an AKTAprime plus chromatographic system (GE Healthcare, USA). Purification includes the following steps: equilibration of the chromatographic column, sample application, column washing, protein elution.

Equilibration of the chromatographic column involves washing with a five-fold volume of base buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.0-7.2). The flow rate was determined by the contact time of the buffer with the sorbent; the contact time was at least 4 min.

Then, the culture liquid containing antibodies was applied to the column; the contact time of the sample with the sorbent was at least 5 min.

The column was washed from unbound proteins, as well as from proteins with a low interaction strength with the sorbent, by sequential washing of the chromoatographic column with the base buffer and the wash buffer (20 mM Tris-HCl, pH 6.8-7.0). The volumes of the base and wash buffers were 3.5 column volumes. The flow rate was determined by the contact time of the buffer with the sorbent; the contact time was at least 3.5 min.

Elution was performed with a five-fold volume of elution buffer (100 mM Glycine-HCl, pH 3.4-3.5). The flow rate was determined by the contact time of the buffer with the sorbent; the contact time was at least 6 min. The resulting samples were neutralized to pH 7.0 using titration buffer (1M Tris). The resulting preparations were analyzed by separation in polyacrylamide gel (PAGE) according to the standard method (Fig. 5). Preparations applied to PAGE: lane 1 - initial culture liquid containing the accumulated antibody, before purification on a chromatographic column, 10 μl; lane 2 — culture liquid that has passed through the chromatographic column and contains cellular proteins not bound to the sorbent, 10 μl; lane 3 - the preparation after washing the column with the equilibration buffer, contains cellular proteins weakly bound to the sorbent, 10 μl; lane 4 — the preparation after washing the column with the washing buffer, contains the remains of unbound proteins, 10 μl; 5 - marker of protein lengths 10-260 kDa; lane 6 — preparation of antibody 15742 after purification, 10 μl. The presented electrophoretogram shows that the obtained antibody is purified from foreign proteins, has a mass of about 50 kDa.

The preparations containing the recombinant antibody were filtered from precipitated proteins using a syringe attachment with a pore diameter of 0.22 μm (TPP, Switzerland). Eluates were concentrated by centrifugation of antibody preparations in concentrators (Sartorius AG, Germany) at 6000 g, 17 ° C. The concentration of antibodies was determined using a device for determining ultra-low concentrations NanoPhotometer NP80 (Implen, Germany) according to the manufacturer's instructions. The control was carried out using separation in PAGE. An example of determining the concentration of a preparation of purified scFv-Fc antibody 15742 obtained from one of the producer clones is shown in FIG. 6. To determine the approximate concentration, preparations of purified scFv-Fc antibody 15742 were diluted 10, 20, 40, 80 times and compared with bovine serum albumin (BSA) at a concentration of 0.025 to 1 μg. Preparations applied to PAGE: lane 1 - preparation of purified antibody 15742 after concentration in a 1-fold dilution (isolation from 1 liter of culture medium), 10 μl; lane 2 - preparation of purified antibody 15742 before concentration in a 10-fold dilution (isolation from 1 L of culture medium), 10 μL; lane 3 - preparation of purified antibody 15742 before concentration in a 20-fold dilution (isolation from 1 L of culture medium), 10 μL; lane 4 - preparation of purified antibody 15742 before concentration in a 40-fold dilution (isolation from 1 L of culture medium), 10 μL; lane 5 — preparation of purified antibody 15742 before concentration in an 80-fold dilution (isolation from 1 L of culture medium), 10 μL; lane 6, protein length marker 10-260 kDa; 7 - BSA - 1 μg; 8 - BSA - 0.1 μg; 9 - BSA - 0.05 μg; 10 - BSA - 0.025 μg. When comparing the concentrations of scFv-Fc antibody 15742 and BSA, it was concluded that the concentration of the target protein obtained from one of the producer clones is approximately 150 mg / L.

Analysis of the culture fluid of various producer clones showed that the production of antibodies in them is uneven. The content of antibodies in the preparations after chromatography and concentration varies from 35 mg to 150.6 mg from one liter of the culture liquid.

Sequence list:

<110> Federal Budgetary Institution of Science "State Scientific Center

virology and biotechnology "Vector" of the Federal Service for Surveillance in

the sphere of consumer protection and human well-being

(FBSI SSC VB "Vector" Rospotrebnadzor)

<120> Universal integration vector pVEAL and recombinant plasmid

pVEAL-15742, providing synthesis and secretion of scFv-Fc antibody ADI-15742

against the Ebola virus in mammalian cells and obtained using

vector pVEAL

<160> SEQ ID NO 2

<210> SEQ ID NO: 1

<211> 10914

<212> PRT

<213> Artificial Sequence

<223> Nucleotide sequence of the universal integration

vector pVEAL

<400> 1

1 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg

61 ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcggt tccctataca

121 gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta

181 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt

241 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat

301 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt

361 aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttca ttggcgccct

421 ccgcgcctac agctcaagcc acatccgaag ggggagggag ccgggagctg cgcgcggggc

481 cgccgggggg aggggtggca ccgcccacgc cgggcggcca cgaagggcgg ggcagcgggc

541 gcgcgcgcgg cggggggagg ggccggcgcc gcgcccgctg ggaattgggg ccctaggggg

601 agggcggagg cgccgacgac cgcggcactt accgttcgcg gcgtggcgcc cggtggtccc

661 caaggggagg gaagggggag gcggggcgag gacagtgacc ggagtctcct cagcggtggc

721 ttttctgctt ggcagcctca gcggctggcg ccaaaaccgg actccgccca cttcctcgcc

781 cgccggtgcg agggtgtgga atcctccaga cgctggggga gggggagttg ggagcttaaa

841 aactagtacc cctttgggac cactttcagc agcgaactct cctgtacacc aggggtcagt

901 tccacagacg cgggccaggg gtgggtcatt gcggcgtgaa caataatttg actagaagtt

961 gattcgggtg tttccggaag gggccgagtc aatccgccga gttggggcac ggaaaacaaa

1021 aagggaaggc tactaagatt tttctggcgg gggttatcat tggcgtaact gcagggacca

1081 cctcccgggt tgagggggct ggatctccag gctgcggatt aagcccctcc cgtcggcgtt

1141 aatttcaaac tgcgcgacgt ttctcacctg ccttcgccaa ggcaggggcc gggaccctat

1201 tccaagaggt agtaactagc aggactctag ccttccgcaa ttcattgagc gcatttacgg

1261 aagtaacgtc gggtactgtc tctggccgca agggtgggag gagtacgcat ttggcgtaag

1321 gtggggcgta gagccttccc gccattggcg gcggataggg cgtttacgcg acggcctgac

1381 gtagcggaag acgccttagt gggggggaag gttctagaaa agcggcggca gcggctctag

1441 cggcagtagc agcagcgccg ggtcccgtgc ggaggtgctc ctcgcagagt tgtttctcca

1501 gcagcggcag ttctcactac agcgccagga cgagtccggt tcgtgttcgt ccgcggagat

1561 ctctctcatc tcgctcggct gcgggaaatc gggctgaagc gactgagtcc gcgatggagg

1621 taacgggttt gaaatcaatg agttattgaa aagggcatgg cgaggccgtt ggcgcctcag

1681 tggaagtcgg ccagccgcct ccgtgggaga gaggcaggaa atcggaccaa ttcagtagca

1741 gtggggctta aggtttatga acggggtctt gagcggaggc ctgagcgtac aaacagcttc

1801 cccaccctca gcctcccggc gccatttccc ttcactgggg gtgggggatg gggagctttc

1861 acatggcgga cgctgccccg ctggggtgaa agtggggcgc ggaggcggga cttcttattc

1921 cctttctaaa gcacgctgct tcgggggcca cggcgtctcc tcggggatct tcaatattgg

1981 ccattagcca tattattcat tggttatata gcataaatca atattggcta ttggccattg

2041 catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg

2101 ccatgttggc attgattatt gactagttat taatagtaat caattacggg gtcattagtt

2161 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga

2221 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca

2281 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca

2341 gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg

2401 cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc

2461 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt

2521 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt

2581 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg cgatcgcccg

2641 ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc

2701 gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg

2761 ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt gactctctta

2821 aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga

2881 caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc

2941 tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac

3001 tcccagttca attacagctc ttaaggctag agtacttaat acgactcact ataggctagc

3061 ctcgagaatt cgtcctgctg cgcacgtggg aagccctggc cccggccgcc accatgatga

3121 ggcccatcgt gctggtgctg ctgttcgcca cctcagcgct ggccggcgag tgcgagtgcg

3181 agtgcgagtc gtacgtacgt acgtacgtac gtacgtatat atatatatat attatatata

3241 ttaatatatg gcgagtgcga gtgcgagtgc gagtcgtacg tacgtacgta cgtacgtacg

3301 tatatatata tatatattat atatattaat atatatatgg atccggagac aaaactcaca

3361 catgcccacc gtgcccagca cctgaactcc tggggggacc gtcagtcttc ctcttccccc

3421 caaaacccaa ggacaccctc tacatcaccc gggaacctga ggtcacatgc gtggtggtgg

3481 acgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc gtggaggtgc

3541 ataatgccaa gacaaagccg cgggaggagc agtacaacag cacgtaccgt gtggtcagcg

3601 tcctcaccgt cctgcaccag gactggctga atggcaagga gtacaagtgc aaggtctcca

3661 acaaagccct cccagccccc atcgcgaaaa ccatctccaa agccaaaggg cagccccgag

3721 aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac caggtcagcc

3781 tgacctgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg gagagcaatg

3841 ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac ggctccttct

3901 tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac gtcttctcat

3961 gctccgtgat gcatgaggct ctgaagttcc actacacgca gaagagcctc tccctgtctc

4021 cgggtaaatg agtcgagcgg ttcccgcccc tctccctccc ccccccctaa cgttactggc

4081 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg

4141 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct

4201 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca

4261 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg

4321 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct

4381 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa

4441 tggctcacct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt

4501 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa

4561 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat

4621 atggccaagt tgaccagtgc cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc

4681 gagttctgga ccgaccggct cgggttctcc cgggacttcg tggaggacga cttcgccggt

4741 gtggtccggg acgacgtgac cctgttcatc agcgcggtcc aggaccaggt ggtgccggac

4801 aacaccctgg cctgggtgtg ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag

4861 gtcgtgtcca cgaacttccg ggacgcctcc gggccggcca tgaccgagat cggcgagcag

4921 ccgtgggggc gggagttcgc cctgcgcgac ccggccggca actgcgtgca cttcgtggcc

4981 gaggagcagg actgagcggc cgcttccctt tagtgagggt taatgcttcg agcagacatg

5041 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt

5101 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa

5161 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggagat gtgggaggtt

5221 ttttaaagca agtaaaacct ctacaaatgt ggtaaaatcc gataaggatc gatccgggct

5281 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg

5341 gcgaatggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag

5401 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt

5461 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt

5521 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg

5581 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt

5641 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt

5701 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca

5761 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttcctgat gcggtatttt

5821 ctccttacgc atctgtgcgg tatttcacac cgcatacgcg gatctgcgca gcaccatggc

5881 ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga aagaaccagc

5941 tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca ggcagaagta

6001 tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag

6061 caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc ccgcccctaa

6121 ctccgcccat cccgccccta actccgccca gttccgccca ttctccgccc catggctgac

6181 taattttttt tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt

6241 agtgaggagg cttttttgga ggccacggcc ggccctccgc gcctacagct caagccacat

6301 ccgaaggggg agggagccgg gagctgcgcg cggggccgcc ggggggaggg gtggcaccgc

6361 ccacgccggg cggccacgaa gggcggggca gcgggcgcgc gcgcggcggg gggaggggcc

6421 ggcgccgcgc ccgctgggaa ttggggccct agggggaggg cggaggcgcc gacgaccgcg

6481 gcacttaccg ttcgcggcgt ggcgcccggt ggtccccaag gggagggaag ggggaggcgg

6541 ggcgaggaca gtgaccggag tctcctcagc ggtggctttt ctgcttggca gcctcagcgg

6601 ctggcgccaa aaccggactc cgcccacttc ctcgcccgcc ggtgcgaggg tgtggaatcc

6661 tccagacgct gggggagggg gagttgggag cttaaaaact agtacccctt tgggaccact

6721 ttcagcagcg aactctcctg tacaccaggg gtcagttcca cagacgcggg ccaggggtgg

6781 gtcattgcgg cgtgaacaat aatttgacta gaagttgatt cgggtgtttc cggaaggggc

6841 cgagtcaatc cgccgagttg gggcacggaa aacaaaaagg gaaggctact aagatttttc

6901 tggcgggggt tatcattggc gtaactgcag ggaccacctc ccgggttgag ggggctggat

6961 ctccaggctg cggattaagc ccctcccgtc ggcgttaatt tcaaactgcg cgacgtttct

7021 cacctgcctt cgccaaggca ggggccggga ccctattcca agaggtagta actagcagga

7081 ctctagcctt ccgcaattca ttgagcgcat ttacggaagt aacgtcgggt actgtctctg

7141 gccgcaaggg tgggaggagt acgcatttgg cgtaaggtgg ggcgtagagc cttcccgcca

7201 ttggcggcgg atagggcgtt tacgcgacgg cctgacgtag cggaagacgc cttagtgggg

7261 gggaaggttc tagaaaagcg gcggcagcgg ctctagcggc agtagcagca gcgccgggtc

7321 ccgtgcggag gtgctcctcg cagagttgtt tctccagcag cggcagttct cactacagcg

7381 ccaggacgag tccggttcgt gttcgtccgc ggagatctct ctcatctcgc tcggctgcgg

7441 gaaatcgggc tgaagcgact gagtccgcga tggaggtaac gggtttgaaa tcaatgagtt

7501 attgaaaagg gcatggcgag gccgttggcg cctcagtgga agtcggccag ccgcctccgt

7561 gggagagagg caggaaatcg gaccaattca gtagcagtgg ggcttaaggt ttatgaacgg

7621 ggtcttgagc ggaggcctga gcgtacaaac agcttcccca ccctcagcct cccggcgcca

7681 tttcccttca ctgggggtgg gggatgggga gctttcacat ggcggacgct gccccgctgg

7741 ggtgaaagtg gggcgcggag gcgggacttc ttattccctt tctaaagcac gctgcttcgg

7801 gggccacggc gtctcctcgg ggatctagct tgtggaaggc tactcgaaat gtttgaccca

7861 agttaaacaa tttaaaggca atgctaccaa atactaattg agtgtatgta aacttctgac

7921 ccactgggaa tgtgatgaaa gaaataaaag ctgaaatgaa tcattctctc tactattatt

7981 ctgatatttc acattcttaa aataaagtgg tgatcctaac tgacctaaga cagggaattt

8041 ttactaggat taaatgtcag gaattgtgaa aaagtgagtt taaatgtatt tggctaaggt

8101 gtatgtaaac ttccgacttc aactgtatag ggttcctcta gctagagacg acctcgggta

8161 cccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag

8221 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc

8281 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc

8341 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa

8401 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg

8461 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg

8521 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag

8581 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac

8641 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga

8701 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt

8761 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc

8821 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc

8881 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta

8941 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat

9001 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca

9061 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct

9121 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt

9181 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct

9241 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc

9301 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa

9361 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta

9421 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc

9481 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat

9541 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta

9601 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt

9661 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt

9721 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg

9781 tgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc

9841 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc

9901 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg

9961 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga

10021 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta

10081 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct

10141 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag

10201 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga

10261 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat

10321 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgc gccctgtagc

10381 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc

10441 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt

10501 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac

10561 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag

10621 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa

10681 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg

10741 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac

10801 aaaatattaa cgcttacaat ttccattcgc cattcaggct gcgcaactgt tgggaagggc

10861 gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctg

<210> SEQ ID NO: 2

<211> 11475

<212> PRT

<213> Artificial Sequence

<223> Nucleotide sequence of the recombinant plasmid pVEAL-15742,

providing the synthesis and secretion of scFv-Fc antibodies ADI-15742 against

the Ebola virus.

<400> 2

1 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg

61 ccagtgagcg cgcgtaatac gactcactat agggcgaatt ggagctcggt tccctataca

121 gttgaagtcg gaagtttaca tacacttaag ttggagtcat taaaactcgt ttttcaacta

181 ctccacaaat ttcttgttaa caaacaatag ttttggcaag tcagttagga catctacttt

241 gtgcatgaca caagtcattt ttccaacaat tgtttacaga cagattattt cacttataat

301 tcactgtatc acaattccag tgggtcagaa gtttacatac actaagttga ctgtgccttt

361 aaacagcttg gaaaattcca gaaaatgatg tcatggcttt agaagcttca ttggcgccct

421 ccgcgcctac agctcaagcc acatccgaag ggggagggag ccgggagctg cgcgcggggc

481 cgccgggggg aggggtggca ccgcccacgc cgggcggcca cgaagggcgg ggcagcgggc

541 gcgcgcgcgg cggggggagg ggccggcgcc gcgcccgctg ggaattgggg ccctaggggg

601 agggcggagg cgccgacgac cgcggcactt accgttcgcg gcgtggcgcc cggtggtccc

661 caaggggagg gaagggggag gcggggcgag gacagtgacc ggagtctcct cagcggtggc

721 ttttctgctt ggcagcctca gcggctggcg ccaaaaccgg actccgccca cttcctcgcc

781 cgccggtgcg agggtgtgga atcctccaga cgctggggga gggggagttg ggagcttaaa

841 aactagtacc cctttgggac cactttcagc agcgaactct cctgtacacc aggggtcagt

901 tccacagacg cgggccaggg gtgggtcatt gcggcgtgaa caataatttg actagaagtt

961 gattcgggtg tttccggaag gggccgagtc aatccgccga gttggggcac ggaaaacaaa

1021 aagggaaggc tactaagatt tttctggcgg gggttatcat tggcgtaact gcagggacca

1081 cctcccgggt tgagggggct ggatctccag gctgcggatt aagcccctcc cgtcggcgtt

1141 aatttcaaac tgcgcgacgt ttctcacctg ccttcgccaa ggcaggggcc gggaccctat

1201 tccaagaggt agtaactagc aggactctag ccttccgcaa ttcattgagc gcatttacgg

1261 aagtaacgtc gggtactgtc tctggccgca agggtgggag gagtacgcat ttggcgtaag

1321 gtggggcgta gagccttccc gccattggcg gcggataggg cgtttacgcg acggcctgac

1381 gtagcggaag acgccttagt gggggggaag gttctagaaa agcggcggca gcggctctag

1441 cggcagtagc agcagcgccg ggtcccgtgc ggaggtgctc ctcgcagagt tgtttctcca

1501 gcagcggcag ttctcactac agcgccagga cgagtccggt tcgtgttcgt ccgcggagat

1561 ctctctcatc tcgctcggct gcgggaaatc gggctgaagc gactgagtcc gcgatggagg

1621 taacgggttt gaaatcaatg agttattgaa aagggcatgg cgaggccgtt ggcgcctcag

1681 tggaagtcgg ccagccgcct ccgtgggaga gaggcaggaa atcggaccaa ttcagtagca

1741 gtggggctta aggtttatga acggggtctt gagcggaggc ctgagcgtac aaacagcttc

1801 cccaccctca gcctcccggc gccatttccc ttcactgggg gtgggggatg gggagctttc

1861 acatggcgga cgctgccccg ctggggtgaa agtggggcgc ggaggcggga cttcttattc

1921 cctttctaaa gcacgctgct tcgggggcca cggcgtctcc tcggggatct tcaatattgg

1981 ccattagcca tattattcat tggttatata gcataaatca atattggcta ttggccattg

2041 catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc aatatgaccg

2101 ccatgttggc attgattatt gactagttat taatagtaat caattacggg gtcattagtt

2161 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga

2221 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca

2281 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca

2341 gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga cggtaaatgg

2401 cccgcctggc attatgccca gtacatgacc ttacgggact ttcctacttg gcagtacatc

2461 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac caatgggcgt

2521 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt

2581 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactg cgatcgcccg

2641 ccccgttgac gcaaatgggc ggtaggcgtg tacggtggga ggtctatata agcagagctc

2701 gtttagtgaa ccgtcagatc actagaagct ttattgcggt agtttatcac agttaaattg

2761 ctaacgcagt cagtgcttct gacacaacag tctcgaactt aagctgcagt gactctctta

2821 aggtagcctt gcagaagttg gtcgtgaggc actgggcagg taagtatcaa ggttacaaga

2881 caggtttaag gagaccaata gaaactgggc ttgtcgagac agagaagact cttgcgtttc

2941 tgataggcac ctattggtct tactgacatc cactttgcct ttctctccac aggtgtccac

3001 tcccagttca attacagctc ttaaggctag agtacttaat acgactcact ataggctagc

3061 ctcgagaatt cgtcctgctg cgcacgtggg aagccctggc cccggccgcc accatgatga

3121 ggcccatcgt gctggtgctg ctgttcgcca cctcagcgct ggccggcgag gtgcagctgg

3181 tggagtccgg gggaggcttg gtacagccgg gggggtccct gagactctcc tgtgcagcct

3241 ctggattcac ctttagcagc tatgccatga gctgggtccg ccaggctcca gggaaggggc

3301 tggagtgggt ctccgaaatt agcggtcttg gtggtagcac atactacgca gactccgcga

3361 agggccggtt caccatctcc agagacaatt ccaagagcac cctgtatctg caaatgaaca

3421 gcctgagagc cgaagacacg gccgtatatt actgtgcgaa agatcatcgc gtttgggcac

3481 ctggatatta ctttgaccac tggggccagg gaaccctggt cactgtgagc tctggtggtg

3541 gtggtagcgg cggcggcggc tctggtggtg gtggttccga tattgtgctg acacagtctc

3601 cttccaccct gtctgcatct gtcggagaca gagtcaccat cacttgccgg gccagtcaga

3661 gtattagtag ctggttggcc tggtatcagc agaaaccagg gaaagcccct aaactcctga

3721 tctatgatgc ctccagtttg gaaagtgggg tcccatcaag gttcagcggc agtggatctg

3781 ggacagagtt cactctcacc atcagcagcc tgcagcctga tgattttgca acttatttct

3841 gccaacagta taataggtcc cccactttcg gcggagggac caaggtggaa atcaagggtg

3901 gatccggaga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac

3961 cgtcagtctt cctcttcccc ccaaaaccca aggacaccct ctacatcacc cgggaacctg

4021 aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt

4081 acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca

4141 gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg

4201 agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgcgaaa accatctcca

4261 aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga

4321 tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg

4381 ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc

4441 tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc

4501 agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgaagttc cactacacgc

4561 agaagagcct ctccctgtct ccgggtaaat gagtcgagcg gttcccgccc ctctccctcc

4621 ccccccccta acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat

4681 atgttatttt ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct

4741 gtcttcttga cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg

4801 ttgaatgtcg tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta

4861 gcgacccttt gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag

4921 ccacgtgtat aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg

4981 atagttgtgg aaagagtcaa atggctcacc tcaagcgtat tcaacaaggg gctgaaggat

5041 gcccagaagg taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca

5101 tgtgtttagt cgaggttaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc

5161 tttgaaaaac acgatgataa tatggccaag ttgaccagtg ccgttccggt gctcaccgcg

5221 cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc

5281 gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc

5341 caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag

5401 ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc

5461 atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc

5521 aactgcgtgc acttcgtggc cgaggagcag gactgagcgg ccgcttccct ttagtgaggg

5581 ttaatgcttc gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga

5641 atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc

5701 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt

5761 cagggggaga tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtaaaatc

5821 cgataaggat cgatccgggc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc

5881 aacagttgcg cagcctgaat ggcgaatgga cgcgccctgt agcggcgcat taagcgcggc

5941 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc

6001 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa

6061 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact

6121 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt

6181 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa

6241 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt

6301 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgcttac

6361 aatttcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgc

6421 ggatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct

6481 tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag

6541 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg

6601 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag

6661 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc

6721 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg

6781 cctctgagct attccagaag tagtgaggag gcttttttgg aggccacggc cggccctccg

6841 cgcctacagc tcaagccaca tccgaagggg gagggagccg ggagctgcgc gcggggccgc

6901 cggggggagg ggtggcaccg cccacgccgg gcggccacga agggcggggc agcgggcgcg

6961 cgcgcggcgg ggggaggggc cggcgccgcg cccgctggga attggggccc tagggggagg

7021 gcggaggcgc cgacgaccgc ggcacttacc gttcgcggcg tggcgcccgg tggtccccaa

7081 ggggagggaa gggggaggcg gggcgaggac agtgaccgga gtctcctcag cggtggcttt

7141 tctgcttggc agcctcagcg gctggcgcca aaaccggact ccgcccactt cctcgcccgc

7201 cggtgcgagg gtgtggaatc ctccagacgc tgggggaggg ggagttggga gcttaaaaac

7261 tagtacccct ttgggaccac tttcagcagc gaactctcct gtacaccagg ggtcagttcc

7321 acagacgcgg gccaggggtg ggtcattgcg gcgtgaacaa taatttgact agaagttgat

7381 tcgggtgttt ccggaagggg ccgagtcaat ccgccgagtt ggggcacgga aaacaaaaag

7441 ggaaggctac taagattttt ctggcggggg ttatcattgg cgtaactgca gggaccacct

7501 cccgggttga gggggctgga tctccaggct gcggattaag cccctcccgt cggcgttaat

7561 ttcaaactgc gcgacgtttc tcacctgcct tcgccaaggc aggggccggg accctattcc

7621 aagaggtagt aactagcagg actctagcct tccgcaattc attgagcgca tttacggaag

7681 taacgtcggg tactgtctct ggccgcaagg gtgggaggag tacgcatttg gcgtaaggtg

7741 gggcgtagag ccttcccgcc attggcggcg gatagggcgt ttacgcgacg gcctgacgta

7801 gcggaagacg ccttagtggg ggggaaggtt ctagaaaagc ggcggcagcg gctctagcgg

7861 cagtagcagc agcgccgggt cccgtgcgga ggtgctcctc gcagagttgt ttctccagca

7921 gcggcagttc tcactacagc gccaggacga gtccggttcg tgttcgtccg cggagatctc

7981 tctcatctcg ctcggctgcg ggaaatcggg ctgaagcgac tgagtccgcg atggaggtaa

8041 cgggtttgaa atcaatgagt tattgaaaag ggcatggcga ggccgttggc gcctcagtgg

8101 aagtcggcca gccgcctccg tgggagagag gcaggaaatc ggaccaattc agtagcagtg

8161 gggcttaagg tttatgaacg gggtcttgag cggaggcctg agcgtacaaa cagcttcccc

8221 accctcagcc tcccggcgcc atttcccttc actgggggtg ggggatgggg agctttcaca

8281 tggcggacgc tgccccgctg gggtgaaagt ggggcgcgga ggcgggactt cttattccct

8341 ttctaaagca cgctgcttcg ggggccacgg cgtctcctcg gggatctagc ttgtggaagg

8401 ctactcgaaa tgtttgaccc aagttaaaca atttaaaggc aatgctacca aatactaatt

8461 gagtgtatgt aaacttctga cccactggga atgtgatgaa agaaataaaa gctgaaatga

8521 atcattctct ctactattat tctgatattt cacattctta aaataaagtg gtgatcctaa

8581 ctgacctaag acagggaatt tttactagga ttaaatgtca ggaattgtga aaaagtgagt

8641 ttaaatgtat ttggctaagg tgtatgtaaa cttccgactt caactgtata gggttcctct

8701 agctagagac gacctcgggt acccagcttt tgttcccttt agtgagggtt aattgcgcgc

8761 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca

8821 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa

8881 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag

8941 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc

9001 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct

9061 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg

9121 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc

9181 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga

9241 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct

9301 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg

9361 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag

9421 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat

9481 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac

9541 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac

9601 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc

9661 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt

9721 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc

9781 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg

9841 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca

9901 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca

9961 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag

10021 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac

10081 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc

10141 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct

10201 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc

10261 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg

10321 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc

10381 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat

10441 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag

10501 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat

10561 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg

10621 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca

10681 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga

10741 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc

10801 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata

10861 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg

10921 ccacctgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc

10981 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt

11041 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc

11101 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt

11161 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt

11221 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt

11281 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa

11341 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttccattcg ccattcaggc

11401 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga

11461 aagggggatg tgctg

Claims (29)

1. Universal integration vector pVEAL, used to create a plasmid genetic construct pVEAL-15742, having the nucleotide sequence SEQ ID No. 1 and containing, in accordance with the physical and genetic map shown in FIG. 1, the following items: - site of the beginning of replication ori (8616-9204); - IR / DR (L / R) sequences, which are part of the SB100 transposase-based system (139-403, 7832-8127); - UCOE sequences preventing chromosomal silencing of the recombinant expression cassette (416-1549, 6272-7820); - CMV Enhancer (2108-2487), CMV promoter (2488-2699) - the strongest most commonly used promoter in gene therapy constructs; - Chimeric intron (2860-2992), which increases the efficiency of the CMV promoter; - the leader peptide V19, which ensures the export of the protein from the cell (3114-3164); - a place for the insertion of variable fragments of the heavy and light chains of the antibody, connected by a peptide linker, at the sites of hydrolysis AfeI (3157), BamHI (3339); - constant chain of human IgG1 antibodies (CH1-CH2-CH3) (3348-4031) with mutations M252Y, S254T, T256E, E333A, H433K / N434F, which increase the half-life of the antibody due to interaction with neonatal receptors; - site of internal landing of the ribosome EMCV IRES (4046-4620); - the Bleo sequence encoding the antibiotic resistance factor bleomycin (4625-4995); - SV40 poly (A) signal sequence required for stabilization of mRNA transcripts (5042-5163); - SV40 promoter (5921-6262), providing autonomous replication in mammalian cells, the combination with the CMV promoter significantly increases the yield of the protein; - antibiotic resistance gene ampicillin AmpR and bacterial promoter of ampicillin resistance gene, allowing preparative plasmid production in E. coli (9375-10235). 2. Plasmid genetic construct pVEAL-15742, providing the synthesis and secretion of the target protein scFv-Fc of the anti-Ebola virus ADI 15742 antibody in mammalian cells, having the nucleotide sequence SEQ ID No. 2 and containing, in accordance with the physical and genetic map shown in FIG. 2, the following items: - site of the beginning of replication ori (8616-9204); - IR / DR (L / R) sequences, which are part of the SB100 transposase-based system (139-403, 8393-8688); - UCOE sequences preventing chromosomal silencing of the recombinant expression cassette (416-1549, 6833-8381); - CMV Enhancer (2108-2487), CMV promoter (2488-2699) - the strongest most commonly used promoter in gene therapy constructs; - Chimeric intron (2860-2992), which increases the efficiency of the CMV promoter; - the leader peptide V19, which ensures the export of the protein from the cell (3114-3164); - ADI-15742-VH, the sequence encoding the variable fragment of the heavy chain of the antibody 15742 (3165-3533); - ADI-15742-VK, the sequence encoding the variable fragment of the light chain of the antibody 15742 (3579-3903); - constant chain of human IgG1 antibodies (CH1-CH2-CH3) (3909-4592) with mutations M252Y, S254T, T256E, E333A, H433K / N434F, which increase the half-life of the antibody due to interaction with neonatal receptors; - site of internal landing of the ribosome EMCV IRES (4607-5181); - the Bleo sequence encoding the antibiotic resistance factor bleomycin (5186-5556); - the SV40 poly (A) signal sequence required for the stabilization of mRNA transcripts (5603-5724); - SV40 promoter (6482-6823), providing autonomous replication in mammalian cells, the combination with the CMV promoter significantly increases the yield of the protein; - the gene for resistance to the antibiotic ampicillin AmpR and the bacterial promoter of the gene for resistance to ampicillin, allowing for preparative production of the plasmid in E. coli (9936-10796).

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