RU2827202C1 - RECOMBINANT PLASMID DNA pVL3-HA/H5 CARRYING INFLUENZA A (H5N8) VIRUS HEMAGGLUTININ GENE, A RECOMBINANT STRAIN OF CHINESE HAMSTER OVARY CELL LINE CHO-K1-H5 AND RECOMBINANT HEMAGGLUTININ OF INFLUENZA A (H5N8) VIRUS, PRODUCED BY CHO-K1-H5 STRAIN AND INTENDED FOR CREATION OF PREVENTIVE PREPARATIONS AND SEROLOGICAL DIAGNOSIS OF INFLUENZA A H5N8 VIRUS - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to creation of a strain of recombinant cell line CHO-K1-H5 producing recombinant hemagglutinin of influenza A (H5N8) virus, which induces a specific immune response against highly pathogenic influenza A (H5N8) virus. RHA/H5 gene has been designed, plasmid DNA pVL3-HA/H5 having the nucleotide sequence SEQ ID NO: 1 5989 bp, molecular weight 3,700,705 Da, containing the target gene coding protein-immunogen HA/H5 SEQ ID NO: 2. Strain of the recombinant cell line CHO-K1-H5 and recombinant hemagglutinin of the influenza A (H5N8) virus, produced by the above cell line strain CHO-K1-H5 and inducing a specific immune response against the highly pathogenic influenza A (H5N8) virus.

EFFECT: recombinant cell line CHO-K1-H5 producing recombinant protein hemagglutinin of influenza A (H5N8) virus, which is capable of inducing a specific protective humoral immune response against influenza A (H5N8) virus.

3 cl, 8 dwg

Description

The invention relates to genetic engineering, biotechnology and medicine. The CHO-K1-H5 strain is described, which is a producer of recombinant hemagglutinin of the influenza A (H5N8) virus, and concerns a method for obtaining a new strain based on CHO-K1 - CHO-K1-H5, which produces a recombinant hemagglutinin protein of the influenza A (H5N8) virus, which can be used in medicine and biotechnology.

Since the end of 2020, outbreaks of highly pathogenic avian influenza A (H5N1) virus subtype (clade 2.3.4.4b) have continued to be recorded worldwide among both wild and domestic birds, resulting in the emergence of new virus variants [1]. This is evidenced by the evolution of the HPAI A (H5N1) subtype recorded in Guangdong Province in 2010 and the recent spread of its genetic reassortant A (H5N8) among domestic and/or wild birds in Japan, the United Kingdom, Romania, Russia and some other countries [2, 3]. Moreover, cases of infection of mammals, in particular humans, have been recorded. Thus, in 2020, the first cases of human infection with a genetic reassortant of the influenza A (H5N8) virus were recorded in the Russian Federation [3, 4].

To effectively combat the highly pathogenic avian influenza A (H5N8) virus, it is necessary to conduct comprehensive studies aimed at both understanding the process underlying constant antigenic variability, evolution, pathogenicity and transmission in animals and humans, and analyzing the structural features of the virus surface proteins that will be used in vaccine development.

It is known that hemagglutinin (HA) is the main active component of vaccines against influenza viruses, inducing protective immunity [5, 6].

The plasmid genetic construct pVEAL3-10H10ch, the strain of the recombinant Chinese hamster ovary cell line CHO-K1-10H10ch and the chimeric antibody 10H10ch against tick-borne encephalitis virus produced by the said strain of the Chinese hamster ovary cell line CHO-K1-10H10ch are known (patent for invention RU 2800471, IPC C12N 15/00, published on 21.07.2023).

However, the above invention is not intended for obtaining the hemagglutinin protein of the influenza A virus.

The closest analogue (prototype) is the hemagglutinin antigen of the avian influenza A virus, the strain of Chinese hamster ovary cells CHO expressing it, the method for producing hemagglutinin and the vaccine based on it and belongs to the field of biotechnology (patent CN 109111508, IPC C12N 15/85, published on September 19, 2018) [9]. The hemagglutinin antigen of the avian influenza virus is the hemagglutinin protein expressed by the sequence presented in SEQ ID NO. 1, and has the advantage of a broad spectrum of action as an antigen, and the hemagglutinin protein is an aprotein expressed by a eukaryotic cell, so that the conformational epitope that loses the antigen can be avoided. The method for obtaining the hemagglutinin antigen of the avian influenza virus and the cell strain expressing the hemagglutinin antigen of the avian influenza virus make it possible to obtain the hemagglutinin protein, which has a broad spectrum and antigenic immunogenicity. The vaccine based on the hemagglutinin antigen of the avian influenza virus has a low production cost and high immunological efficiency.

After immunization of SPF chickens aged 3 to 4 weeks, the H5 subtype antigen can be detected on the 14th day to determine the geometric mean titer (GMT) of HI antibody (GMT) of at least 1:64, which is superior to conventional vaccines.

However, when designing the protein, only the most conservative epitopes of the hemagglutinin of influenza A (H5N1) virus strains sequenced between 2013 and 2018 were used.

The technical result of the claimed invention is the expansion of the spectrum of prophylactic agents against the influenza A (H5N8) virus and its serological diagnostics by creating a more modern producer strain based on Chinese hamster ovary cells - CHO-K1-H5, producing recombinant hemagglutinin of the influenza A (H5N8) virus, which retains its antigenic properties and has immunogenic and protective properties.

The technical result is achieved by obtaining the integrative plasmid DNA pVL3-HA/H5, having the nucleotide sequence SEQ ID NO 1 of 5989 bp in size, a molecular weight of 3,700,705 Da, containing the target gene encoding the hemagglutinin protein of the influenza A virus, providing the expression and secretion of recombinant hemagglutinin with a trimerizing domain of fibritin of the T4 bacteriophage of the influenza A virus (H5N8) in mammalian cells and containing, in accordance with the physical and genetic map, the following elements:

- ori - the replication origin site, with coordinates from 5381 to 5968 bp;

- enhancer with coordinates from 369 to 733 bp. and the CMV promoter at coordinates 734 to 937 bp;

- 5'SB and 3'SB are the binding sites of SB transposase, with coordinates from 88 to 368 bp and from 4039 to 4316 bp, respectively;

- rHA/H5 is a nucleotide sequence encoding the hemagglutinin protein of the influenza A (H5N8) virus with the trimerizing domain of the fibrin of the bacteriophage T4, having coordinates from 1026 to 2657 bp;

- EMCV IRES - internal ribosome entry site, with coordinates from 2733 to 3307 bp;

- PuroR is a sequence encoding a resistance factor to the antibiotic puromycin, with coordinates from 3320 to 3919 bp;

- SV40 poly(A) signal - a sequence required for the stabilization of mRNA transcripts due to polyadenylation, with coordinates from 3954 to 4075 bp;

- KanR is a gene for resistance to the antibiotic kanamycin, which allows the plasmid to be produced in E. coli and has coordinates from 4560 to 5375 bp.

The integrative plasmid DNA pVL3-HA/H5 in complex with the plasmid SB100 (Addgene) allows for efficient integration of the target gene into the genome of CHO-K1 cells, providing a high and stable level of synthesis of recombinant hemagglutinin in the form of trimers.

The technical result is also achieved by creating a recombinant strain of the Chinese hamster ovary cell line CHO-K1-H5, containing the plasmid DNA pVL3-HA/H5 according to claim 1 with an integrated nucleotide sequence rHA/H5 and ensuring the expression and secretion of the hemagglutinin of the influenza A virus (H5N8) in the form of trimers.

The specified technical result is also achieved by obtaining a recombinant hemagglutinin of the influenza A (H5N8) virus with a trimerizing domain of the fibritin of the bacteriophage T4, produced by the strain of the Chinese hamster ovary cell line CHO-K1-H5 according to claim 2, having the amino acid sequence SEQ ID NO: 2 and intended for the creation of prophylactic drugs against the influenza A (H5N8) virus and its serological diagnostics.

When designing the influenza A (H5N8) virus hemagglutinin protein with the trimerizing domain of bacteriophage T4 fibritin, the transmembrane and cytoplasmic domains were removed [5]; the cleavage site between HA1 (head domain) and HA2 (stalk) PLREKRRKRG was replaced by PQRETRG to preserve the uncleaved protein; the T4 trimerizing domain was added at the C-terminus to stabilize the trimer structure [7, 8] and a poly-His-tag for subsequent purification [9], having the amino acid sequence SEQ ID NO:2.

The invention is illustrated by the following graphic materials.

Fig. 1. Physical and genetic map of plasmid pVL3-HA/H5.

Fig. 2. Electropherogram of the separation of purified recombinant hemagglutinin rHA/H5, obtained in the producer strain CHO-K1-H5, in PAGE under denaturing and native conditions:

Lane 1 - molecular mass marker Precision Plus Protein Dual Color Standards (Bio-rad, USA);

Lane 2 - purified preparation of recombinant hemagglutinin rHA/H5 under denaturing conditions;

Lane 3 - purified preparation of recombinant hemagglutinin rHA/H5 under native conditions;

Fig. 3. Western blot analysis of purified recombinant hemagglutinin H5 obtained in the producer strain CHO-K1-H5:

Lane 1 - molecular mass marker Precision Plus Protein Dual Color Standards (Bio-rad, USA);

Lane 2 - purified recombinant hemagglutinin rHA/H5 preparation detected using serum from a ferret infected with influenza A (H5N8) virus.

Fig. 4. Antibody titers in the serum of immunized animals. Recombinant protein rHA/H5 was used as an antigen for sorption in ELISA. rHA/H5 is a group of animals immunized with recombinant hemagglutinin rHA/H5; H5N8 virus is a group of animals immunized with inactivated influenza A virus (H5N8); intact animals are a group of animals that have not been subjected to any manipulations.

Fig. 5. Virus-neutralizing activity of immune sera against influenza A/turkey/Stavropol/320-01/2020 (H5N8) virus. Recombinant rHA/H5 protein - group of animals with recombinant rHA/H5 hemagglutinin; inactiv. influenza A (H5N8) virus - group of animals immunized with inactivated influenza A (H5N8) virus; intact animals - group of animals that have not been subjected to any manipulations.

Fig. 6. Survival curves obtained in different groups of immunized animals after infection with the influenza virus strain A/Astrakhan/3212/2020 (H5N8). Recombinant protein rHA/H5 is a group of animals immunized with recombinant hemagglutinin rHA/H5; inactivated influenza A (H5N8) virus is a group of animals immunized with inactivated influenza A (H5N8) virus; intact animals are a group of animals that have not been subjected to any manipulations.

Fig. 7. Nucleotide sequence of plasmid DNA pVL3-HA/H5.

Fig. 8. Amino acid sequence of recombinant hemagglutinin of influenza A virus (H5N8).

For a better understanding of the essence of the proposed invention, examples of its implementation are given below.

Example 1. Construction of the pVL3-HA/H5 plasmid, providing the synthesis and secretion of the hemagglutinin of the influenza A virus (H5N8) in the form of trimers

Based on the pVL3 vector, the integration plasmid pVL3-HA/H5 (Fig. 1) containing the hemagglutinin sequence of the influenza A (H5N8) virus was obtained. To construct the recombinant protein, the sequence of the gene encoding the full-length hemagglutinin H5 protein was used, the source of which was the A/turkey/Stavropol/320-01/2020 virus (EPI_ISL_1114749).

When designing the amino acid sequence of the recombinant hemagglutinin, the transmembrane and cytoplasmic domains were removed from the natural sequence [5]; the cleavage site between HA1 (head domain) and HA2 (stalk) PLREKRRKRG was replaced by PQRETRG to preserve the uncleaved protein; a T4 trimerizing domain was added at the C-terminus to stabilize the trimer structure [7, 8] and a poly-His-tag for subsequent purification [9]. As a result, the amino acid sequence of the influenza A (H5N8) virus hemagglutinin protein with the trimerizing domain of bacteriophage T4 fibrin, designated as rHA/H5, was designed. The rHA/H5 gene encoding rHA/H5 was synthesized and inserted into the pVL3 vector at the AsuNHI and SalI sites. As a result, a DNA construct encoding rHA/H5 was obtained, designated as pVL3-HA/H5 (Fig. 1) and having the sequence SEQ ID NO: 1 (Fig. 7).

Example 2. Obtaining the CHO-K1-H5 strain producing hemagglutinin of the influenza A (H5N8) virus

The CHO-K1-H5 strain was obtained on the basis of the CHO-K1 cell line using the developed pVL3-HA/H5 construct. CHO-K1 cells were grown in an incubator at 5% _CO2 and 70% humidity. When the monolayer density reached 80%, the cells were transfected with a mixture of pVL3-HA/H5 and pCMV(CAT)T7-SB100 plasmids taken in a 10:1 ratio using Lipofectamine 3000 (ThermoFisher, USA) in a 12-well plate according to the manufacturer's instructions. Forty-eight hours after transfection, the selective antibiotic puromycin was added at a concentration of 10 μg/ml, and selection of resistant cells in which integration had occurred was performed. The resulting polyclonal culture was then seeded into a 96-well flat-bottomed culture plate at a concentration of 1-2 cells/200 μl in DMEM/F12 medium (Servicebio) containing 10% fetal bovine serum (HiMedia) and 10 μg/ml puromycin (Invivogen) and grown until a monolayer was reached. As a result, CHO-K1-H5 monocultures were obtained, which were cryo-frozen.

Characteristics of the recombinant strain CHO-K1-H5.

Morphology: spindle-shaped and epithelial-like cells with round nuclei containing 1 to 2 nucleoli.

Cultivation method: monolayer.

Culture medium: DMEM/F-12 nutrient medium (1:1) - 90%, fetal bovine blood serum - 10%.

Cultivation temperature: 37°C.

Seeding concentration: 100 thousand cells in 1 ml.

Removal method: 0.25% trypsin (1/3) and 0.02% estradiol (2/3).

Sieving ratio: 1:3.

Passaging frequency: 3-4 days.

Cryopreservation conditions: nutrient medium DMEM/F-12 (1:1) - 50%, fetal bovine blood serum - 40%, DMSO - 10%.

Freezing mode: at a temperature of 4°C - 1 hour, minus 80°C - 12 hours, minus 196°C.

Storage conditions: in cryotubes in the amount of 5 pieces, stored in liquid nitrogen at a temperature of minus 196°C.

Passage number in liquid nitrogen: 4.

Viability after cryopreservation: 85-90%.

Marker feature: the presence in the culture medium of the recombinant hemagglutinin protein rHA/H5 of the influenza A (H5N8) virus, ~75 kDa in size, confirmed using protein electrophoresis and immunoblotting.

Application area: biotechnology.

Example 3. Production and purification of recombinant hemagglutinin of influenza A (H5N8) virus

The monoclonal culture of CHO-K1-H5 was grown in roller flasks at 37°C in DMEM/F-12 medium (Servicebio) supplemented with 5% FBS (Himedia) for 7 days. The culture medium was then collected and the protein was purified.

Purification of recombinant rHA/H5 hemagglutinin was performed by metal chelate chromatography on a Ni-NTA column (Qiagen). Before loading the sample, the culture medium was centrifuged to remove cellular debris for 15 min at 5000 rpm at 4°C. After that, the supernatant was diluted 1:1 with binding buffer (20 mM Na2HPO4, 0.5 M NaCl, pH 7.4) and loaded onto the column. Then the column was washed with wash buffer (20 mM Na2HPO4, 0.5 M NaCl, 30 mM imidazole, pH 7.4). The target protein was eluted with elution buffer (20 mM Na2HPO4, 0.5 M NaCl, 500 mM imidazole, pH 7.4). Purification of the target protein was assessed by electrophoresis in 12% PAGE followed by fixation and staining with Coomassie G250. Fractions containing the target protein were pooled and dialyzed against PBS (Neofroxx). The protein preparation was then quantified by the Bradford method using a spectrophotometer by measuring the optical density at 260/280 nm.

The antigenicity of purified rHA/H5 hemagglutinin was confirmed by Western blot analysis. Western blot analysis was performed using the SNAP i.d. 2.0 system (Millipore) according to the manufacturer's protocol. The primary antibody was serum from a ferret infected with influenza A (H5N8) virus (dilution 1:100), and the secondary antibody was precipitated immunoglobulins from mouse serum (dilution 1:3000) and goat anti-mouse immunoglobulin G conjugated with alkaline phosphatase (1:5000). Visualization of the immune complex was performed using the 1-Step™ NBT/BCIP substrate (Thermo Scientific).

As a result, it was confirmed that the hemagglutinin of the influenza A virus (H5N8) with the amino acid sequence SEQ ID NO: 2 (Fig. 8) after purification and subsequent dialysis remains in a soluble form in the form of trimers and retains its antigenic properties (Fig. 2, Fig. 3).

Example 4. Immunogenicity of recombinant hemagglutinin of influenza A (H5N8) virus.

The work with animals was carried out in accordance with the "Guide for the Care and Use of Laboratory Animals". The protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the State Research Center of Virology and Biotechnology "Vector" (BEC Protocol No. 1 dated 03/21/2023).

To assess the immunogenicity of the recombinant hemagglutinin of the influenza A (H5N8) virus, female BALB/c mice weighing 16-18 g were used. The animals were divided into three groups (10 individuals per group) and immunized intramuscularly twice with a three-week interval. The first group was administered 50 μg of purified recombinant hemagglutinin rHA/H5, resuspended in physiological solution, in the presence of complete Freund's adjuvant (1st immunization) and incomplete Freund's adjuvant (2nd immunization) (Sigma) in a total volume of 100 μl. The second group was administered inactivated influenza A (H5N8) virus intramuscularly also in combination with complete (1st immunization) and incomplete Freund's adjuvant (2nd immunization) in a total volume of 100 μl as a positive control. The third group consisted of intact animals. Fourteen days after the second immunization, blood was collected from the retro-orbital sinus. The serum was separated from cellular elements by centrifugation (9000 g, 15 min), heated for 30 min at 56°C and tested for the presence of antibodies specifically binding to the rHA/H5 protein in ELISA. The samples were also analyzed for their neutralizing activity.

For ELISA, rHA/H5 was adsorbed onto 96-well plates (Corning, USA) at a concentration of 1 μg/ml in PBS overnight at 4°C. The plates were washed three times and incubated for 1.5 h at room temperature with 1% casein in PBS with 0.05% Tween 20 to block nonspecific protein binding. The plates were then incubated with three-fold serial dilutions of mouse serum for 1 h at room temperature; after washing with PBST, horseradish peroxidase-conjugated rabbit anti-mouse IgG (Sigma, USA) diluted 1:3000 was added and incubated for 1 h at room temperature. After the final wash, the plates were developed by adding TMB substrate (Amresco, USA). The reaction was stopped with 1N HCl and analyzed at 450 nm on a Varioskan LUX multi-mode microplate reader (Thermo Fisher Scientific, USA).

According to the ELISA results at the end point of the experiment, the average titers of HA/H5-specific antibodies in the group of animals immunized with the recombinant rHA/H5 protein were 1:500,000 (Fig. 4).

The neutralizing properties of blood sera were determined by the inhibition of the cytopathic effect (CPE) of the virus in cell culture in vitro [5]. Two days after the start of incubation of MDCK cells with a mixture of serial dilutions of sera and 100 TCID50/well of the A/turkey/Stavropol/320-01/2020 strain, the cells were stained with a crystal violet solution (1.3 g of the dye was dissolved in 50 ml of 96% ethyl alcohol, brought to 700 ml with distilled water and 300 ml of 40% formalin solution was added) and analyzed using an Agilent BioTek Cytation 5 multi-mode cell visualization reader (Thermo fisher scientific). The neutralization titer was taken as the value of the highest serum dilution that achieved >50% virus neutralization, which corresponds to >50% viable cells.

As a result, it was found that the sera of animals immunized with recombinant hemagglutinin rHA/H5 are capable of neutralizing the live influenza virus strain A/turkey/Stavropol/320-01/2020 with an average titer of 1:2260 (Fig. 5).

Example 5. Study of the ability of recombinant hemagglutinin of influenza A (H5N8) virus to protect against infection with A/turkey/Stavropol/320-01/2020 (H5N8) virus after immunization of animals.

The ability of recombinant hemagglutinin rHA/H5 to protect against virus infection was analyzed using BALB/c mice, which are susceptible to infection with the influenza A/Astrakhan/3212/2020 (H5N8) virus. Fourteen days after the second immunization, the mice were infected intranasally with a 20 MLD50 dose of the influenza A/Astrakhan/3212/2020 (H5N8) virus strain homologous to the studied strain. Manipulations to infect mice were performed under anesthesia with a mixture of Zoletil100 and Xyla. The animals were observed daily after infection for 14 days after infection, while clinical signs as an indicator of the disease (activity, appetite) and death were monitored.

Based on the results of the experiment, it was established that the protective effect in groups of mice immunized with recombinant hemagglutinin rHA/H5 was 100% (Fig. 6).

Sources of patent and scientific and technical information

1. Mahmoud S.N. et al. Immunogenicity and Cross-Protective Efficacy Induced by an Inactivated Recombinant Avian Influenza A/H5N1 (Clade 2.3. 4.4 b) Vaccine against Co-Circulating Influenza A/H5Nx Viruses // Vaccines. - 2023. - T. 11. - No. 9.

2. Zhao K. et al. Characterization of three H5N5 and one H5N8 highly pathogenic avian influenza viruses in China // Veterinary microbiology. - 2013. - T. 163. - No. 3-4. - pp. 351-357.

3. World Health Organization. Human infection with avian influenza A (H5N8)-the Russian Federation. Disease Out-break News. [Electronic resource]. 2021. URL: https://www.who.int/emergencies/disease-outbreak-news/item/2021 - DON313 (accessed 10/25/2023).

4. Pyankova O.G. et al. Isolation of clade 2.3. 4.4 b A (H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm, Russia, December 2020 // Eurosurveillance. - 2021. - T. 26. - No. 24.

5. Yamada S., Yasuhara A., Kawaoka Y. Soluble recombinant hemagglutinin protein of H1N1pdm09 influenza virus detects cross-protection against a lethal H5N1 challenge in mice // Frontiers in microbiology. - 2019. - T. 10.

6. Chen T.H. et al. Recombinant hemagglutinin produced from Chinese Hamster Ovary (CHO) stable cell clones and a PELC/CpG combination adjuvant for H7N9 subunit vaccine development // Vaccine. - 2019. - T. 37. - No. 47. - pp. 6933-6941.

7. Lu Y., Welsh J.P., Swartz J.R. Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines // Proceedings of the National Academy of Sciences. - 2014. - T. 111. - No. 1. - pp. 125-130.

8. Ecker J.W. et al. High-yield expression and purification of recombinant influenza virus proteins from stably-transfected mammalian cell lines // Vaccines. - 2020. - T. 8. - No. 3. - P. 462.

9. Bornhorst J.A., Falke J.J. [16] Purification of proteins using polyhistidine affinity tags // Methods in enzymology. - Academic Press, 2000. - T. 326. - P. 245-254.

10. Patent CN 109111508, IPC C12N 15/85, published 19.09.2018 (prototype).

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aaaatgaatacacagtttgaggccgtgggcagagagttcaataatctggagcgtaggatagaaaatctca

ataagaagatggaggatgggttcctagacgtttggacctacaacgctgaattgttagtgttgatggagaa

cgagcgcaccctcgacttccacgacagcaatgtgaagaacctctatgataaagtgagacttcaattaaga

gataatgcaaaggagcttggcaacggatgttttgagttttatcataagtgtgacaatgaatgcatggagt

cagtccgtaacggcacctacgattaccctcagtatagtgaagagggctacatccctgaggcccctagaga

tggccaggcctatgtgagaaaggatggcgagtgggtgctgctgtctacctttctgcatcaccaccatcat

caccatcaccaccactaagtcgaccgagcggttcccgcccctctccctcccccccccctaacgttactgg

ccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtctttt

ggcaatgtgagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcg

ccaaaggaatgcaaggtctgttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaac

aacgtctgtagcgaccctttgcaggcagcggaaccccccacctggcgacaggtgcctctgcggccaaaag

ccacgtgtataagatacacctgcaaaggcggcacaaccccagtgccacgttgtgagttggatagttgtgg

aaagagtcaaatggctcacctcaagcgtattcaacaaggggctgaaggatgcccagaaggtaccccattg

tatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaacgtctag

gccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatatggccacaaccatgaccg

agtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgc

gttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctg

caagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcgg

tggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggc

cgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaag

gagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccg

tcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccg

caacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgc

acctggtgcatgacccgcaagcccggtgcctgattcgcatatgggttaatgcttcgagcagacatgataa

gatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttg

tgatgctattgctttatttgtaaccattataagctgcaataaacaagttcctcgacctctagctagagct

actcgggaccccttaccgaaacatcgccgcattctgcagaggagtcgagtgtatgtaaacttctgaccca

ctgggaatgtgatgaaagaaataaaagctgaaatgaatcattctctctactattattctgatatttcaca

ttcttaaaataaagtggtgatcctaactgacctaagacagggaatttttactaggattaaatgtcaggaa

ttgtgaaaaagtgagtttaaatgtatttggctaaggtgtatgtaaacttccgacttcaactgtataggga

tccgctcaatactgaccatttaaatcatacctgacctccatagcagaaagtcaaaagcctccgaccggag

gcttttgacttgatcggcacgtaagaggttccaactttcaccataatgaaataagatcactaccgggcgt

attttttgagttatcgagattttcaggagctaaggaagctaaaatgagccatattcaacgggaaacgtct

tgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatg

tcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaaca

tggcaaaggtagcgttgccaatgatgttacagatgagatggtcaggctaaactggctgacggaatttatg

cctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatcccag

ggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagt

gttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacggcgatcgcgtatttcgtctg

gctcaggcgcaatcacgaatgaataacggtttggttggtgcgagtgattttgatgacgagcgtaatggct

ggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactca

tggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacga

gtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttcat

tacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcacttgat

gctcgatgagtttttctaatgagggcccaaatgtaatcacctggctcaccttcgggtgggcctttctgcg

ttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgatgctcaagtcagaggt

ggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgt

tccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagc

tcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccg

ttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatc

gccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttg

aagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagtta

cctcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtt

tgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgattttctac</INSDSeq_

sequence>

</INSDSeq>

</SequenceData>

<SequenceData sequenceIDNumber="2">

<INSDSeq>

<INSDSeq_length>543</INSDSeq_length>

<INSDSeq_moltype>AA</INSDSeq_moltype>

<INSDSeq_division>PAT</INSDSeq_division>

<INSDSeq_feature-table>

<INSDFeature>

<INSDFeature_key>source</INSDFeature_key>

<INSDFeature_location>1..543</INSDFeature_location>

<INSDFeature_quals>

<INSDQualifier>

<INSDQualifier_name>mol_type</INSDQualifier_name>

<INSDQualifier_value>protein</INSDQualifier_value>

</INSDQualifier>

<INSDQualifier id="q4">

<INSDQualifier_name>organism</INSDQualifier_name>

<INSDQualifier_value>synthetic construct</INSDQualifier_value>

</INSDQualifier>

</INSDFeature_quals>

</INSDFeature>

</INSDSeq_feature-table>

<INSDSeq_sequence>MENIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDIL

EKTHNGKLCDLNGVKPLILKDCSVAGWLLGNPMCDEFIRVPEWSYIVERANPANDLCYPGSLNDYEELKH

LLSRINHFEKILIIPKSSWPNHETSLGVSAACPYQGAPSFFRNVVWLIKKNDAYPTIKISYNNTNREDLL

ILWGIHHSNNAEEQTNLYKNPTTYISVGTSTLNQRLVPKIATRSQVNGQRGRMDFFWTILKPDDAIHFES

NGNFIAPEYAYKIVKKGDSTIMKSGVEYGHCNTKCQTPVGAINSSMPFHNIHPLTIGECPKYVKSNKLVL

ATGLRNSPLREKGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDK

MNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRD

NAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEGYIPEAPRDGQAYVRKDGEWVLLSTFLHHHHHH

HHHH</INSDSeq_sequence>

</INSDSeq>

</SequenceData>

</ST26SequenceListing>

<---

Claims (11)

1. Recombinant plasmid DNA pVL3-HA/H5, having the nucleotide sequence SEQ ID NO: 1, 5989 bp in size, molecular weight of 3,700,705 Da, containing the target gene encoding the hemagglutinin protein of the influenza A virus, providing expression and secretion of recombinant hemagglutinin with the trimerizing domain of the fibritin of the T4 bacteriophage of the influenza A virus (H5N8) in mammalian cells and containing, in accordance with the physical and genetic map, the following elements: - ori - the replication origin site, with coordinates from 5381 to 5968 bp; - enhancer with coordinates from 369 to 733 bp. and the CMV promoter at coordinates 734 to 937 bp; - 5'SB and 3'SB are the binding sites of SB transposase, with coordinates from 88 to 368 bp and from 4039 to 4316 bp, respectively; - rHA/H5 is a nucleotide sequence encoding the hemagglutinin protein of the influenza A (H5N8) virus with the trimerizing domain of the fibrin of the bacteriophage T4, having coordinates from 1026 to 2657 bp; - EMCV IRES - internal ribosome entry site, with coordinates from 2733 to 3307 bp; - PuroR is a sequence encoding a resistance factor to the antibiotic puromycin, with coordinates from 3320 to 3919 bp; - SV40 poly(A) signal - a sequence required for the stabilization of mRNA transcripts due to polyadenylation, with coordinates from 3954 to 4075 bp; - KanR is a gene for resistance to the antibiotic kanamycin, which allows the plasmid to be produced in E. coli and has coordinates from 4560 to 5375 bp. 2. A recombinant strain of the Chinese hamster ovary cell line CHO-K1-H5, containing the plasmid DNA pVL3-HA/H5 according to claim 1 with an integrated nucleotide sequence rHA/H5 and producing the hemagglutinin of the influenza A (H5N8) virus. 3. Recombinant hemagglutinin of the influenza A (H5N8) virus, produced by the strain of the Chinese hamster ovary cell line CHO-K1-H5 according to claim 2, having the amino acid sequence SEQ ID NO: 2 and intended for the creation of prophylactic drugs and serological diagnostics of the influenza A (H5N8) virus.