RU2761378C1 - Gene encoding the open reading frame of the m segment of cchf and recombinant structure ensuring expression of structural glycoproteins of the crimean-congo hemorrhagic fever virus - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a gene encoding structural glycoproteins of the Crimean-Congo hemorrhagic fever virus. Also described is a recombinant plasmid ensuring expression of said gene by means of a cytomegalovirus (CMV) promoter, as well as integration thereof into the genome of mammalian cells. The invention can be used in biotechnology, molecular biology, genetic engineering, and medicine. The described technical result is achieved by creating a gene encoding the open reading frame of the M segment of the CCHF virus, represented by the sequence SEQ ID NO: 1 with a length of 5052 bps. Created is a pSB3delta-GPC plasmid with a size of 11,495 bps, containing, according to the physical and genetic map shown in fig. 4, a target gene encoding the open reading frame of the M segment of the CCHF virus with an amino acid sequence SEQ ID NO: 2 with a length of 1,683 a.a.

EFFECT: creation of a recombinant plasmid ensuring integration and expression of structural glycoproteins of the Crimean-Congo hemorrhagic fever virus.

2 cl, 7 dwg, 4 tbl

Description

The invention relates to a gene encoding the structural glycoproteins of the Crimean-Congo hemorrhagic fever virus and a recombinant plasmid providing the expression of this gene due to the cytomegalovirus (CMV) promoter, as well as their integration into the genome of mammalian cells due to inverted repeats flanking the sequence of the gene of interest, promoter elements and a selective marker, and can be used in biotechnology, molecular biology, genetic engineering, and medicine, in particular, to obtain a vaccine.

Crimean-Congo hemorrhagic fever is an especially dangerous natural focal vector-borne disease caused by the Crimean-Congo hemorrhagic fever virus (CCHF), one of the most widespread arbovirus infections in the world, and is also a recurrent infection endemic to the south of the European part of Russia. The CCHF virus belongs to the Nairoviridae family, order Bunyavirales. The genome of the CCHF virus consists of three segments: small (S), medium (M) and large (L), represented by single-stranded RNA of negative polarity [1].

The carriers of the virus are ixodid ticks of the genus Hyalomma, and wild mammals are the hosts and reservoir in nature. The area of distribution of the CCHF virus practically coincides with the area of distribution of ticks of the genus Hualomma and covers Africa and southern Eurasia. For more than 30 countries, the incidence of CCHF has been identified or the presence of the CCHF virus in ticks has been shown [10].

There is currently no approved vaccine, but there are many vaccine formulations that include subunit antigens, genetically modified plants, viral vectors and DNA vaccines expressing CCHF antigens, virus-like particles (VLP), messenger RNA (mRNA) preparations, and inactivated viral particles [7].

A patent solution is known (WO 2020123989, IPC A61K 39/12, publ. 06/18/2020) [11], where a variant of replicon viral particles was proposed, containing all viral proteins, S- and L-segments of the genome, but not containing full-size M-segment. The production of viral particles in cell culture was achieved by transfection with pCAGGS plasmids containing all three reading frames and with minigenomic plasmids pT7 containing full-length S and L segments. To increase the titer, the authors additionally propagated replicon viral particles in Huh7 cell culture, also transfected with pCAGGS-GPC-Oman.

The closest analogue (prototype) is a solution to the patent (US 9109199, IPC A61K 39/12, publ. 08/18/2015) [12], which describes a method of obtaining replicon particles of Bunyaviruses, including the Rift Valley fever virus and CCHF, by trans -complementation of structural glycoproteins Gc and Gn both in the presence and in the absence of NSm coding regions. Trans-complementation was achieved by transient expression of complete viral glycoproteins in BHK-21 cell culture with the pCAGGS plasmid encoding the open reading frame of the M-segment of the CCHF virus, or by using a viral vector encoding the open reading frame of the M-segment of the CCHF virus. The patent discloses an approach to the design of replicon particles of bunyaviruses in order to study the immune response, develop diagnostic tools for bunyavirus infections, and also study the properties of viruses outside the premises of the BSL-4 class.

The disadvantages of the above solutions are the use of additional plasmids or a viral vector, which would provide the production of viral glycoproteins, which may lead to a decrease in the titer of replicon particles, as well as the loss of the ability of the cell line to express glycoproteins. Additionally, when the M-segment is excluded from the viral particle, the T-cell response decreases, since intracellular synthesis of the main structural antigen is not provided. In addition, this solution is not feasible for the serial production of vaccine preparations, since during passaging of suspension cell cultures, their constant transfection with an expression plasmid encoding viral glycoproteins is required.

Thus, there is a need in the art for the development of new approaches that provide more stable expression of viral genes and their use for the development of candidate vaccine preparations.

Disclosure of invention

The technical result of the claimed invention is the creation of a recombinant plasmid that provides integration and stable expression of structural glycoproteins of the Crimean-Congo hemorrhagic fever virus and does not have the disadvantages of the above analogs and prototype.

The specified technical result is achieved by creating a gene encoding an open reading frame of the M-segment of the CCHF virus, which is represented by the sequence SEQ ID NO: 1 5052 bp in length.

This technical result is also achieved by creating a recombinant plasmid pSB3delta-GPC, which has a size of 11495 bp. and contains, in accordance with the physical and genetic map shown in FIG. 4, a target gene encoding a precursor protein (open reading frame of the M-segment of the CCHF virus) having the amino acid sequence SEQ ID NO: 2 1683 aa in length, and consists of the following elements:

• β-lactamase gene (coordinates from 10442 to 11303 bp) under the control of a promoter (coordinates from 11304 to 11409 bp), as a genetic marker that determines ampicillin resistance of E. coli cells;

• the point of origin of replication ori of the plasmid vector pUC (coordinates from 9683 to 10272 bp)

• 5'-ITR and 3'-ITR inverted repeats with coordinates from 17 to 241 bp. and from 9215 to 9439 p.n. accordingly, ensure the integration of the plasmid region enclosed between them into the genome of mammalian cells using Sleeping Beauty (SB) transposase;

• promoter and enhancer of human cytomegalovirus (CMV) - provides gene expression according to claim 1 in mammalian cells (coordinates from 855 to 1438 bp);

• himeric intron - chimeric intron providing efficient transcription, stability and transport of the transcript from the cell nucleus (coordinates from 1574 to 1706 bp);

• T7 promoter - the nucleotide sequence of the promoter of bacteriophage T7, required for in vitro transcription (coordinates from 1751 to 1769 bp);

• Glycoprotein precursor - open reading frame of the gene encoding the structural glycoproteins of the CCHF virus (coordinates from 1783 to 6834 bp);

• IRES2 - internal site of encephalomyelitis virus ribosome entry, providing expression of the puromycin resistance gene (coordinates from 6879 to 7465 bp);

• PuroR - puromycin resistance gene encoding N-acetyl transferase (coordinates from 7466 to 8068 bp);

• bGH poly (A) signal - polyadenelation signal (coordinates from 8099 to 8323 bp).

Significant differences from the prototype, ensuring the achievement of the technical result, are:

1. The promoter IE-element of human cytomegalovirus (CMV) is required to obtain stably expressing cell lines;

2. The resulting recombinant plasmid is an integration vector that contains ITRs flanking the sequence of the gene of interest, promoter elements and selectable marker. These ITRs sequences are recognized by the Sleeping Beauty (SB) transposase, which is used to integrate into the cell genome;

3. The gene for resistance to the antibiotic puromycin allows for selection of stably expressing cell transformants;

4. Viral genes for structural glycoproteins were obtained from an epidemiologically significant CCHF virus strain.

The essence of the claimed invention is illustrated by drawings. FIG. 1 shows the scheme of amplification of the open reading frame of the M-segment of the CCHF virus. FIG. 2 shows an electrophoretogram in 1.5% agarose gel of fragments of open reading frames of M- and L-segments of the CCHF virus, amplified on a cDNA template. FIG. 3 shows an electrophoretogram in 1.5% agarose gel of overlap extension PCR products obtained by combining amplicons, where M is a 1 Kb M12 DNA marker (SibEnzyme, Russia), GPC is an open reading frame of an M segment, RdRp is an open reading frame of an L segment. ... FIG. 4 shows a schematic diagram of the construction of the recombinant plasmid pSB3delta-GPC. FIG. 5 shows an electrophoretogram in 1% agarose gel of DNA fragments after hydrolysis of plasmids pSB3delta-GPC and pSB3delta-RdRp with restriction endonucleases XhoI and MluI, where M is a 1 Kb M12 DNA marker (SibEnzyme, Russia), GPC is pSB3delta DNA hydrolyzed XhoI - MluI, RdRp - pSB3delta-RdRp DNA hydrolyzed by XhoI - MluI enzymes. FIG. 6 shows the populations of HEK293-A cell lines two (A), five (B) days, and seven (C) days after transfection with plasmids pSB100X and pSB3delta-GPC. FIG. 7 shows the electrophoretic separation of the amplification products of the internal region of the M-segment of the CCHF virus, where M is a 1 kb M12 DNA length marker (SibEnzyme, Russia); 1-5 - amplicons obtained using a solution of isolated RNA / DNA with a DNA concentration of 50 ng to 0.005 ng; 6-10 - amplicons obtained using a sample after reverse transcription with a DNA concentration from 50 ng to 0.005 ng; K- - negative control sample containing PNR-mixture without DNA template.

Implementation of the invention. The first stage in the creation of the target plasmid was the choice of a method for expressing the target gene in cell cultures. For this, the Sleeping Beauty transposon system was chosen, which allows highly efficient insertion into the genome of DNA fragments of various lengths limited by recognizable special sequences - the so-called inverted terminal repeats (ITR). The cassette intended for insertion into the genome and limited by ITR is delivered into the cell as part of a plasmid vector. With the simultaneous delivery of the vector and the expression of transposase in the cell, the cassette is excised from the vector and inserted into an arbitrary place in the genome of the host cell. Thus, the stable transfection of cell cultures is highly efficiently carried out [8]. The use of cell lines stably expressing viral proteins can be an advantage in the development of candidate DNA vaccines and VLPs against the CCHF virus and other viruses of the order Bunyavirales.

The regions encoding the structural glycoproteins (open reading frame of the M segment) of the CCHF virus were selected as the target gene. This choice is due to the fact that viral glycoproteins (Gc and Gn, VP38) contain many immunogenic epitopes and are used in most approaches to induce both humoral and cellular responses [2, 9]. It was shown that candidate vaccine preparations containing these sites in their composition protected mice during challenge [3-5].

Thus, the present invention can be used for trans-complementation of the M-segment when creating replicon particles of bunyaviruses.

Example 1. Obtaining cDNA copies of the M-segment of the CCHF virus

The selection of primers (Table 1) was carried out using the SnapGene v.3.2.1 and Oligo7 software, using the Ast199 isolate from the M-segment of the CCHF virus as a reference (GenBank sequences: KX056051, KX056050). The synthesis of oligonucleotide primers was carried out by the biotechnological company "Evrogen" (Moscow). Restriction sites in oligonucleotides are indicated by underline and bold.

1.1. Isolation of viral RNA and staging of the reverse transcription reaction

Isolation of total RNA was performed from vaccinated material (infected monolayer of Vero cell culture in AVL lysis buffer). For isolation, a commercial QIAamp Viral RNA Mini Kit (Qiagen, Germany) was used according to the manufacturer's recommendations. The eluate was stored at minus 20 ° C; in the next stage of the work, we used it for staging reverse transcription.

The RNAscribe RT kit (Biolabmix, Russia) was used to obtain the first DNA strand on the viral genomic RNA template. To carry out the reaction, a test tube with a mixture of the necessary components was placed in a Thermal Cycler C1000 thermocycler (BioRad, USA) and the temperature-time profile recommended by the manufacturer was used.

1.2. PCR amplification of cDNA fragments encoding glycoproteins of the CCHF virus

The regions were amplified by PCR using specific primers (Table 1) according to FIG. 1. For amplification, high-precision Phusion High-Fidelity polymerase (NEB, USA) was used, the reaction was carried out according to the manufacturer's recommendations. The reaction for each fragment was carried out in duplicate using a Thermal Cycler C1000 amplifier with a given temperature-time profile (Table 2)

1.3. Isolation of DNA from an agarose gel. After PNR, DNA amplification products were purified by electrophoresis in 1.5% agarose gel in tris-acetate buffer (TAE × 1), stained with ethidium bromide solution for 10 min, and developed using a UV transilluminator "UV Transilluminator 2000" (BioRad, USA). After visualization under UV radiation, DNA fragments of the required length, separated in an agarose gel (Fig. 2), were excised and isolated from the gel using the QIAquick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's recommendations.

1.4. Combining fragments using overtap-extension PCR

The concentration of isolated DNA fragments was measured using a NanoDrop One (Thermo Scientific, USA). After that, the fragments were mixed in equimolar amounts and the first stage (overlap) of the synthesis of cDNA copies encoding the ORF of the M segment using Phusion High-Fidelity polymerase (NEB, USA) was carried out using the temperature-time profile presented in Table 3. The second stage (extension) was carried out using terminal primers containing the restriction sites Smal and Mlul (Table 1) with a given temperature-time profile (Table 4).

Purification of the products of DNA amplification using agarose gel electrophoresis was carried out by the method described earlier. After visualization under UV radiation, DNA fragments of the required length, separated in an agarose gel (Fig. 3), were excised and isolated from the gel in a similar manner.

Example 2 Obtaining the pSB3detta-GPC integration construct carrying the open reading frame of the M-segment of the CCHF virus

2.1. Cloning of PCR products carrying the open reading frame of the M-segment of the CCHF virus into the pSB3delta plasmid

To clone the genes of structural glycoproteins into the pSB3delta vector, enzymatic hydrolysis of the plasmid vector and PCR products was carried out using restriction endonucleases SmaI and MluI (NEB, USA) with attached buffers. The reaction mixture was prepared in accordance with the enzyme activity and DNA concentration. The reaction conditions and the duration of the enzymatic hydrolysis of DNA were selected in accordance with the manufacturer's instructions.

Enzyme-treated PCR products and the pSB3 delta vector were separated by electrophoresis in 1.5% agarose gel followed by isolation using a QIAquick Gel Extraction Kit (Qiagen, Germany) according to the manufacturer's recommendations.

The ligation reaction was carried out for 30 minutes at 22 ° C using a mixture of each of the PCR products, the pSB3delta vector plasmid in equimolar amounts and the T4 phage DNA ligase (ThermoFisher, USA) in the reaction buffer supplied with the commercial kit. The resulting DNA construct pSB3delta-GPC (Fig. 4) was used to transform the culture of competent cells of E. coli strain NEB Stable (NEB, USA). Transformants were selected using a standard PCR screening procedure for the inner portion of each insert.

2.2. Obtaining the pSB3delta-GPC plasmid construct and confirming its structure by restriction analysis. Using a bacteriological loop, the colony was transferred from the Petri dish into a test tube containing 3.5 ml of SOB liquid nutrient medium with the selective antibiotic ampicillin (100 μg / ml). Then the tubes were placed in an incubator shaker for 16-18 hours at 170 rpm, 30 ° C. The resulting bacterial suspension was used to isolate plasmid DNA using the QIAprep Spin Miniprep Kit (Qiagen, Germany) according to the manufacturer's recommendations.

To confirm the structure of the obtained plasmid construct, the pSB3delta-GPC plasmid construct was hydrolyzed using restriction endonucleases XhoI and MluI. According to the theoretically calculated sequence, hydrolysis of the pSB3delta-GPC plasmid with the above enzymes should result in fragments of 5471 bp, 3195 bp, 1440 bp in length. and 1389 bp.

FIG. 5 shows an electropherogram of DNA fragments after hydrolysis of plasmid pSB3delta-GPC with restriction endonucleases XhoI and MluI in 1% agarose gel, where:

M - DNA marker 1 Kb M12 (SibEnzyme, Russia)

GPC - pSB3delta-GPC DNA hydrolyzed by XhoI - MluI enzymes

RdRp - pSB3delta-RdRp DNA hydrolyzed by XhoI - MluI enzymes

From the data presented in FIG. 5 that the mobility of the pSB3delta-GPC and pSB3delta-RdRp plasmid hydrolyzate fragments in 1% agarose gel relative to the molecular weight markers corresponds to the mobility of the theoretically calculated fragments.

2.3. Plasmid structure confirmation by Sanger sequencing

To confirm the correct nucleotide sequence of the obtained samples, the Sanger sequencing method was used. To set up the reaction, the BrilliantDye Terminator (v3.1) Cycle Sequencing kit (Nimagen, Netherlands) was used, the samples were purified through Sephadex G-50 Superfine (Cytiva, Sweden) by the column method according to the manufacturer's recommendations, after which they were transferred for capillary gel electrophoresis on automatic sequencer 3500xl Applied Biosystems (USA). The resulting chromatograms were analyzed using SnapGene 3.2.1 software.

Example 3. Lipofection of the HEK293-A cell line with pSB100X / pSB3delta-GPC plasmids and confirmation of gene expression. To create cell lines stably expressing the GPC polyprotein gene, a continuous cell line HEK293-A was chosen. Lipofectamine 3000 kit (Thermo Fisher Scientific, USA) was used for lipofection. The amount taken for transfection of plasmid DNA in total was 3 μg for one well, with a ratio of 1 part of plasmid pSB 100X to 9 parts of plasmid pSB3delta-GPC. The lipofection mixture was prepared according to the manufacturer's recommendations. Before transfection, the cells were plated on a 6-well culture plate, 2 ml of cell suspension with a concentration of 100 thousand cells / ml per well in a nutrient growth medium DMEM / F-12 with 7% fetal bovine serum. The plate was incubated to obtain a cell monolayer with a confluence of 70-90%.

In a 6-well culture plate containing a cell line monolayer, the DMEM / F-12 nutrient medium was replaced with a medium with a reduced serum content OptiMEM (2 ml per well), after which 250 μl of the resulting mixture of the DNA-lipid complex was added to 3 wells spreading the mixture over the entire surface. As a control, 3 wells with a cell monolayer were left without adding a DNA-lipid complex. After incubation for two days at 37 ° C in a CO ₂ incubator, the conditioned culture medium with a reduced serum content OptiMEM was replaced with DMEM / F-12 medium supplemented with the selective antibiotic puromycin at a concentration of 5 μg / ml. In one of three wells with a monolayer of cells that were not transfected, a culture medium was added without the addition of an antibiotic to monitor the state of the cell population. Next, incubation was carried out at 37 ° C in a CO ₂ incubator with a change of nutrient medium every 2-3 days until a monolayer of transfected cells with a confluence of 70-90% was obtained (Fig. 6).

To confirm the expression of the gene encoding the structural glycoproteins of the CCHF virus, RNA and DNA were isolated from the cell suspension of the transgenic line HEK293-A using the RIBO-prep reagent kit (Helicon, Russia). Part of the solution of purified RNA / DNA was digested with DNase I (NEB, USA), free of RNases, and used to obtain cDNA by reverse transcription with oligonucleotide primers F-CCHFV_inner and R-CCHFV_inner (Table 1) corresponding to the inner region of the M-segment (size 1541 bp). The amount of DNA in the solution of isolated RNA / DNA and the sample after reverse transcription was measured on a NanoDrop One spectrophotometer (Thermo ScientiHc, USA) and PCR was performed with 6 points of 10-fold dilution of 50 ng DNA contained in a solution of purified RNA / DNA and in a sample with cDNA ... Based on the results of electrophoretic separation in agarose gel of the amplification products of the fragment, it can be concluded that the cells of the transgenic line HEK293-A used for the isolation of RNA and DNA contained in the genome the sequence of the gene for structural glycoproteins of the CCHF virus. The presence of RNA in an amount sufficient for successful production of reverse transcription with specific oligonucleotide primers suggests stable expression of this gene in the resulting cell line (Fig. 7), which confirms the claimed technical result.

Sources of patent and scientific and technical information

1. Fields, Bernard N; Knipe, David M; Howley P.M. Fields virology 6th ed. (2013) // Book. 2013. p. 2664.

2. Goedhals D., Paweska J.T., Burt F.J. Long-lived CD8 + T cell responses following Crimean-Congo haemorrhagic fever virus infection // PLoS Neglected Tropical Diseases. 2017. No. 12 (11). C. e0006149.

3. Hinkula J. [et al.]. Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and / or Virus-Like Particles Induces Protection and Survival in Challenged Mice // Journal of Virology. 2017. No. 10 (91).

4. Rodriguez S.E. [and etc.]. Vesicular Stomatitis Virus-Based Vaccine Protects Mice against Crimean-Congo Hemorrhagic Fever // Scientific Reports. 2019. No. 1 (9). C. 1-13.

5. Spengler J. R. [et al.]. Heterologous protection against Crimean-Congo hemorrhagic fever in mice after a single dose of replicon particle vaccine // Antiviral Research. 2019. (170). C. 104573.

6. Sun Y. [et al.]. Bunyavirales ribonucleoproteins: the viral replication and transcription machinery // Critical Reviews in Microbiology. 2018. No. 5 (44). S. 522-540.

7. Tipih T., Burt F.J. Crimean-congo hemorrhagic fever virus: Advances in vaccine development // BioResearch Open Access. 2020. No. 1 (9). S. 137-150.

8. Tschorn N., Berg K., Stitz J. Transposon vector-mediated stable gene transfer for the accelerated establishment of recombinant mammalian cell pools allowing for high-yield production of biologies // Biotechnology Letters. 2020. No. 7 (42). S. 1103-1112.

9. Zhang J. [et al.]. Fine mapping epitope on glycoprotein Gc from Crimean-Congo hemorrhagic fever virus // Comparative Immunology, Microbiology and Infectious Diseases. 2019. (67). S. 24-31.

10. Kulichenko A.H. [and etc.]. Crimean hemorrhagic fever in Eurasia in the XXI century: epidemiological aspects // Epidemiol. and inf. bol. Act. question 2012. (3). S. 42-53.

11. WO 2020123989, IPC A61K 39/12, publ. 06/18/2020 (analogue).

12. US 9109199, IPC A61K 39/12, publ. 08/18/2015 (prototype).

APPENDIX

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Sequence listing

<110> Federal budgetary institution of science "State scientific

Center for Virology and Biotechnology "Vector" of the Federal Service for

supervision of consumer protection and human well-being

(FBUN SSC VB "Vector" Rospotrebnadzor)

<120> The gene encoding the open reading frame of the M-segment CCHF,

and a recombinant construct that provides the expression of structural

glycoproteins of the Crimean-Congo virus of hemorrhagic fever

<160> 2

<210> SEQ ID NO: 1

<211> 5052 bp

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence of an artificial gene encoding

structural glycoproteins of the CCHF virus.

<400> 1

ATGCATACAC TCTTGGTGTG CTTCATTCTT TACCTACAGC TGTCGGGTTT AGGAGGAGCT 60

CATGGACAGT CAAACGCAAC TGAACACAAT GGAACAAACA CCACCACAGT ACCCGGCACA 120

AGTCAAAGCC CCAAGCCACC CGCGAGCACC ACTCTACCAC ACACACCAGA ATCCTCCACC 180

GTCAAACTCA CCACACCAAC AAGCGAAACA GAAGGCTCAG GGGAAACGAC CCCACCCCCA 240

AACACCACCC AGGGCCTGCC CCTACCAGGG ACCACACCAG AACGCTCCGC AACAACCGCC 300

ACAAGCACGT CAAGCACAGA CAACATGAAT TCCACCACAC AGGTGACCGA TAACACCCCC 360

ACATCAACAG TCAGCATAAG TCCATCCAGC AGCTCCAGCA TGCCATCCAC ATCACAAGGC 420

ATACACCATC CCGTAAGGAG TCTATTGTCA GTCTCGAGCC CTAAGACAGC AACAACACCG 480

ACACCAATGA GCCCTGGAGA GATGAGCCCG GAAACCAGCA GTCAGCACTC AGCCATGAGC 540

AGAGCGCCAA CCCTCCACAC AGCAACCCAG GTCTCCACTG AGAACACCGA CCGCAGCACA 600

CCTAGACAAT CTGAGTCCTC AGTACAGCCG GCAACCCAAA GCCCAATAAC TTCCCCAGCG 660

CAAACAATCC TTCTGATGAG TGCTGCTCCT AAAGCTATTC AGGACATGCA TCCCAGCCCA 720

ACAAACAGGT CCAAGAGGAA CCTTGAGGTG GAAATAATTT TAACTCTGTC TCAAGGTCTA 780

AAGAAATATT ACGGTAAAAT ACTGAAGCTT CTGCACCTTA CCTTGGAAGA GGATACTGAA 840

GGCCTGTTAG AATGGTGTAA GAGAAACCTT GGGTCAAATT GTGATGATGA CTTCTTTCAA 900

AAGAGAATAG AAGAATTCTT CATAACTGGT GAGGGCTACT TCAATGAAGT CCTACAATTT 960

AAAACACTAA GCACACTAAG CCCCACGGAG CCGTCTCATG TCAGGCTACC AACAGCGGAG 1020

CCCTTCAAAT CTTACTTCGC TAAGGGCTTC CTTTCAATAG ATTCGGGTTA CTTCTCTGCC 1080

AAATGTTATC CAAGATCATC CACTTCAGGG CTTCAGCTGA TCAATGTCAC CCAACACCCA 1140

GCTAGGATAG CAGAAACACC TGGACCCAAA ACAACGAGTC TGAAAACCAT CAACTGTATC 1200

AACCTAAGAG CATCAGTCTT CAAAGAACAC AGAGAAGTTG AGATCAATGT GCTTCTTCCT 1260

CAGATTGCAG TCAACCTCTC AAACTGCCAT GTTGTGATCA ACTCACATGT CTGTGATTAC 1320

TCTTTAGACA CTGACGGGCC AGTGAGGCTT CCTCGCATTC ACCATGAAGG TACTTTCATA 1380

CCAGGAACTT ATAAAATAGT GATAGACAGG AAAAATAAGC TAAATGACCG ATGCTCGCTA 1440

GTCACTAACT GTGTGATAAA GGGAAGAGAG GTTCGTAAGG GCCAATCAGT GCTGAGGCAG 1500

TACAAAACAG AAATTAAGAT AGGCAAGACA TCAACAGGCT CTAGGAAACT ACTGTCCGAA 1560

GAGCCAGGTG ACGACTGCAT ATCAAGAACT CAGCTATTGA AGACAGAAAC AGCAGAGATC 1620

CATGACGACA ACTATGGTGG TCCAGGTGAC AAAATCACTA TCTGCAATGG TTCAACCATT 1680

GTAGATCAGA GACTGGGCAG TGAACTAGGG TGTTATACCA TCAACAGGGT GAAGTCATTC 1740

AAGCTATGCG AAAACAGTGC CACAGGGAAA ACCTGTGAGA TAGACAGCAC TCCGGTCAAG 1800

TGCAGGCAAG GTTTTTGTTT GAAAATCACC CAAGAGGGAA GGGGCCACGT AAAATTATCC 1860

AGGGGCTCAG AGGTTGTTTT GGATGCTTGC GACTCGAGCT GTGAAATAAT GATACCTAAA 1920

GGCACTGGAG ATATCCTAGT AGACTGTTCA GGTGGACAGC AACATTTCTT AAAAGACAAC 1980

TTGATTGACC TAGGGTGCCC TCATATCCCG CTATTGGGTA AAATGGCCAT TTACATTTGC 2040

AGAATGTCAA ATCATCCCAG GACAACCATG GCTTTCCTCT TCTGGTTCAG TTTTGGCTAT 2100

GTGATCACCT GTATATTTTG CAAGGCTCTT TTTTATTCAT TGATAATTAT TGGGACACTA 2160

GGGAAAAAAA TCAAGCAATA TAGGGAGCTA AAACCTCAAA CTTGCACCAT ATGTGAGACA 2220

GCTCCTGTCA ATGCAATAGA TGCTGAAATG CATGACCTCA ACTGTAGTTA CAATATATGC 2280

CCTTATTGTG CATCAAGACT GACCTCTGAT GGGCTGGCTA GGCATGTGAC ACAATGCCCT 2340

AAGCGAAAGG AGAAAGTTGA GGAGACTGAA CTGTATTTGA ACCTGGAAAG AATTCCTTGG 2400

ATAGTCAGAA AGCTATTGCA AGTGTCAGAG TCCACTGGTG TGGCATTGAA ACGAAGCAGT 2460

TGGCTGATTG TGCTGCTTGT GTTACTCACA GTCTCATTGT CACCGGTCCA ATCAGCACCT 2520

GTTGGTCATG GTAAGACAAT CGAAACATAT CAGACCAGAG AGGGATTCAC AAGTATCTGT 2580

CTCTTTATGT TAGGAAGCAT CCTATTCATA GTTTCTTGCC TGGTAAAGGG GCTGGTTGAC 2640

AGTGTCAGTG ACAGCTTCTT CCCCGGCCTG TCTGTCTGTA AGACATGCTC TATTGGCAGT 2700

ATAAATGGCT TTGAAATTGA ATCGCATAAA TGCTACTGTA GTTTATTTTG CTGCCCCTAC 2760

TGTAGGCACT GCTCTGCTGA CAGAGAAATT CACCAGTTGC ACTTGAGTAT CTGCAAAAAA 2820

AGAAAAACGG GGAGCAATGT CATGCTGGCT GTCTGCAAGC GCATGTGCTT TAAGGCAACC 2880

ATAGAAGCAA GCAGAAGAGC CCTGCTCATC CGAAGCATTA TCAATACCAC TTTTGTAATG 2940

TGCATTCTAA CATTAGCAAT CTGTGTTGTT AGCACCTCTG CAGTAGAGAT GGAAGACCTT 3000

CCAGCAGGCA CTTGGGAGAG AGAGGAAGAC CTAACAAATT TCTGCCATCA GGAATGCCAG 3060

GTGACCGAGA CGGAATGCCT CTGTCCATAC GAAGCTCTTG TGCTCAGGAA ACCCCTTTTC 3120

TTAGACAGTA TAGTTAAAGG TATGAAAAAT TTGCTAAACT CAACAAGCTT AGAAACAAGC 3180

TTATCAATTG AGGCACCATG GGGAGCAATA AATGTCCAGT CAACCTTTAA ACCAACAGTA 3240

TCGACTGCAA ACATAGCACT CAGTTGGAGC TCAGTAGAGC ACAGAGGCAA CAAGATCTTG 3300

GTCACAGGCA GGTCTGAATC AATTATGAAA CTAAAGGAAA GGACAGGAGT CAGCTGGGAT 3360

CTAGGCGTAG AAGATGCCTC TGAATCCAAA TTACTTACCG TCTCTATCAT GGATTTGTCA 3420

CAAATGTATT CCCCTGTTTT TGAGTACTTA TCAGGAGATA GACAGGTTGA AGAATGGCCC 3480

AAGGCAACCT GCACAGGTGA TTGCCCAGAA AGGTGTGGCT GCACATCATC AACCTGCTTG 3540

CATAAAGAAT GGCCTCATTC AAGAAACTGG AGATGTAATC CCACTTGGTG CTGGGGAGTA 3600

GGAACCGGCT GCACATGCTG CGGAGTGGAT GTAAAAGACC TTTTTACAGA CCACATGTTT 3660

ATCAAATGGA AGGTTGAATA TATAAAAACC GAAGCCATAG TATGTGTTGA GCTCACCAGC 3720

CAAGAAAGAC AGTGCAGTCT GATTGAGGCA GGTACTAGAT TCAACTTGGG TCCTGTGACT 3780

ATCACCCTGT CTGAGCCAAG AAACATTCAG CAGAAGCTTC CCCCTGAGAT CATTACGCTG 3840

CATCCCAAAA TTGAAGAAGG CTTCTTTGAC TTGATGCATG TGCAAAAAGT GTTATCTGCA 3900

AGTACAGTGT GTAAACTGCA GAGTTGCACA CATGGTATAC CAGGAGACCT ACAAATTTAC 3960

CACATTGGCA ACTTGTTGAA AGGGGATAGA GTAAACGGCC ATCTAATTCA CAAAATTGAA 4020

CCACATTTTA ACACCTCTTG GATGTCCTGG GATGGTTGTG ACCTAGACTA CTATTGCAAC 4080

ATGGGGGATT GGCCTTCTTG CACATACACA GGAGTGACCC AACATAATCA TGCTGCATTT 4140

GTAAACTTGC TTAACATTGA AACTGATTAC ACGAAAACCT TCCACTTCCA CTCTAAAAGA 4200

GTCACAGCAC ATGGAGACAC ACCACAATTA GACTTAAAAG CAAGGCCGAC CTACGGTGCA 4260

GGTGAGATCA CTGTGCTGGT TGAGGTTGCT GACATGGAGT TGCATACCAA AAAAGTTGAG 4320

ATATCAGGCT TGAAATTTGC AAGTCTGACT TGTACGGGTT GCTATGCTTG TAGCTCTGGC 4380

ATCTCCTGTA AAGTCAGAAT TCATGTAGAT GAACCAGATG AGCTCACAGT GCATGTAAAG 4440

AGCAGTGATC CAGATGTGGT TGCAGCTAGC ACAAGCCTTA TGGCAAGGAA GCTTGAATTT 4500

GGGACAGATA GCACATTCAA GGCATTTTCA GCTATGCCCA AAACCTCTTT ATGCTTTTAC 4560

ATTGTGGAGA GGGAATACTG CAAGAGCTGC AGTGAAGATG ATACAAAAAA ATGTGTTGAC 4620

ACAAAACTTG AGCAGCCACA GAGCATACTA ATTGAGCACA AAGGGACGAT AATTGGAAAG 4680

CAGAATGATA CTTGCACAGC CAAGGCAAGT TGTTGGCTAG AGTCAGTAAA GAGTTTTTTT 4740

TATGGATTGA AGAATATGCT TGGCAGTGTT TTTGGTAGTT TTTTCGTAGG CATTTTGTTA 4800

TTCCTTGCCC CTTTCGTCTT GTTGGTGCTT TTCTTTATGT TTGGATGGAA AATCTTGTTT 4860

TGCTTTAAAT GTTGCAGGAG GACCAGAGGC TTGTTTAAGT ATAGACACCT CAAAGATGAC 4920

GAGGAAACAG GCTACAGAAG GATTATTGAA AGACTGAACA GCAAAAAAGG AAAAAACAGA 4980

CTGCTTGATG GTGAGAGGTT GGCTGATAGG AAAATTGCTG AGCTGTTCTC TACAAAAACA 5040

CACATTGGCT AA 5052

<210> SEQ ID NO: 2

<211> 1683 a.k.o.

<212> PRT

<213> Artificial Sequence

<220>

<223> Amino acid sequence of structural glycoproteins

CCHF virus

<400> 2

MHTLLVCFIL YLQLSGLGGA HGQSNATEHN GTNTTTVPGT SQSPKPPAST TLPHTPESST 60

VKLTTPTSET EGSGETTPPP NTTQGLPLPG TTPERSATTA TSTSSTDNMN STTQVTDNTP 120

TSTVSISPSS SSSMPSTSQG IHHPVRSLLS VSSPKTATTP TPMSPGEMSP ETSSQHSAMS 180

RAPTLHTATQ VSTENTDRST PRQSESSVQP ATQSPITSPA QTILLMSAAP KAIQDMHPSP 240

TNRSKRNLEV EIILTLSQGL KKYYGKILKL LHLTLEEDTE GLLEWCKRNL GSNCDDDFFQ 300

KRIEEFFITG EGYFNEVLQF KTLSTLSPTE PSHVRLPTAE PFKSYFAKGF LSIDSGYFSA 360

KCYPRSSTSG LQLINVTQHP ARIAETPGPK TTSLKTINCI NLRASVFKEH REVEINVLLP 420

QIAVNLSNCH VVINSHVCDY SLDTDGPVRL PRIHHEGTFI PGTYKIVIDR KNKLNDRCSL 480

VTNCVIKGRE VRKGQSVLRQ YKTEIKIGKT STGSRKLLSE EPGDDCISRT QLLKTETAEI 540

HDDNYGGPGD KITICNGSTI VDQRLGSELG CYTINRVKSF KLCENSATGK TCEIDSTPVK 600

CRQGFCLKIT QEGRGHVKLS RGSEVVLDAC DSSCEIMIPK GTGDILVDCS GGQQHFLKDN 660

LIDLGCPHIP LLGKMAIYIC RMSNHPRTTM AFLFWFSFGY VITCIFCKAL FYSLIIIGTL 720

GKKIKQYREL KPQTCTICET APVNAIDAEM HDLNCSYNIC PYCASRLTSD GLARHVTQCP 780

KRKEKVEETE LYLNLERIPW IVRKLLQVSE STGVALKRSS WLIVLLVLLT VSLSPVQSAP 840

VGHGKTIETY QTREGFTSIC LFMLGSILFI VSCLVKGLVD SVSDSFFPGL SVCKTCSIGS 900

INGFEIESHK CYCSLFCCPY CRHCSADREI HQLHLSICKK RKTGSNVMLA VCKRMCFKAT 960

IEASRRALLI RSIINTTFVM CILTLAICVV STSAVEMEDL PAGTWEREED LTNFCHQECQ 1020

VTETECLCPY EALVLRKPLF LDSIVKGMKN LLNSTSLETS LSIEAPWGAI NVQSTFKPTV 1080

STANIALSWS SVEHRGNKIL VTGRSESIMK LKERTGVSWD LGVEDASESK LLTVSIMDLS 1140

QMYSPVFEYL SGDRQVEEWP KATCTGDCPE RCGCTSSTCL HKEWPHSRNW RCNPTWCWGV 1200

GTGCTCCGVD VKDLFTDHMF IKWKVEYIKT EAIVCVELTS QERQCSLIEA GTRFNLGPVT 1260

ITLSEPRNIQ QKLPPEIITL HPKIEEGFFD LMHVQKVLSA STVCKLQSCT HGIPGDLQIY 1320

HIGNLLKGDR VNGHLIHKIE PHFNTSWMSW DGCDLDYYCN MGDWPSCTYT GVTQHNHAAF 1380

VNLLNIETDY TKTFHFHSKR VTAHGDTPQL DLKARPTYGA GEITVLVEVA DMELHTKKVE 1440

ISGLKFASLT CTGCYACSSG ISCKVRIHVD EPDELTVHVK SSDPDVVAAS TSLMARKLEF 1500

GTDSTFKAFS AMPKTSLCFY IVEREYCKSC SEDDTKKCVD TKLEQPQSIL IEHKGTIIGK 1560

QNDTCTAKAS CWLESVKSFF YGLKNMLGSV FGSFFVGILL FLAPFVLLVL FFMFGWKILF 1620

CFKCCRRTRG LFKYRHLKDD EETGYRRIIE RLNSKKGKNR LLDGERLADR KIAELFSTKT 1680

HIG * 1683

<---

Claims (12)

1. The gene encoding the structural glycoproteins of the CCHF virus having a nucleotide sequence of SEQ ID NO: 1 5052 bp in length. 2. The recombinant plasmid pSB3delta-GPC, intended for integration and expression of the gene for structural glycoproteins of the CCHF virus in mammalian cells, has a size of 11495 bp. and contains, in accordance with the physical and genetic map shown in FIG. 4, the target gene according to claim 1, encoding a precursor protein, which is an open reading frame of the M-segment of the CCHF virus, having the amino acid sequence of SEQ ID NO: 2 in length of 1683 aa, and consists of the following elements: • β-lactamase gene (coordinates from 10442 to 11303 bp) under the control of a promoter (coordinates from 11304 to 11409 bp), as a genetic marker that determines ampicillin resistance of E. coli cells; • the origin of replication ori of the plasmid vector pUC (coordinates from 9683 to 10272 bp); • 5'-ITR and 3'-ITR inverted repeats with coordinates from 17 to 241 bp. and from 9215 to 9439 p.n. accordingly, they provide the integration of the plasmid region, enclosed between them, into the genome of mammalian cells using Sleeping Beauty (SB) transposase; • CMV promoter and enhancer - ensures the expression of the gene according to claim 1 in mammalian cells (coordinates from 855 to 1438 bp); • chimeric intron - chimeric intron providing efficient transcription, stability and transport of the transcript from the cell nucleus (coordinates from 1574 to 1706 bp); • T7 promoter - nucleotide sequence of the promoter of bacteriophage T7, required for in vitro transcription (coordinates from 1751 to 1769 bp); • Glycoprotein precursor - open reading frame of the gene encoding the structural glycoproteins of the CCHF virus according to claim 1 (coordinates from 1783 to 6834 bp); • IRES2 - internal site of encephalomyelitis virus ribosome entry, providing expression of the puromycin resistance gene (coordinates from 6879 to 7465 bp); • PuroR - puromycin resistance gene encoding N-acetyl transferase (coordinates from 7466 to 8068 bp); • bGH poly (A) signal - polyadenelation signal (coordinates from 8099 to 8323 bp).

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