EA031404B1 - Vaccine for the prophylaxis and treatment of rotavirus infection with a fusion protein as active agent (variants) - Google Patents

Abstract

The group of inventions relates to molecular biology, biotechnology and medicine and is intended for the prophylaxis and treatment of rotavirus infection. Proposed is a vaccine against rotavirus infection which is based on a fusion protein (SEQ ID NO: 1, SEQ ID NO: 2), consisting of immunogenic epitopes of the VP6 and VP8 proteins linked by a flexible bridge (variants). Also proposed is a vaccine against rotavirus infection which is based on a fusion protein (SEQ ID NO: 3 - SEQ ID NO: 6), consisting of immunogenic epitopes of the VP6 and VP8 proteins and an adjuvant, namely flagellin fragments, linked by flexible bridges (variants). The fusion protein - the active ingredient of vaccines - is produced in prokaryote and eukaryote cells using recombinant DNA technology and various purification methods. The proposed vaccines are shown to be immunologically multivalent against rotavirus groups A, B and C and to have a prophylactic and therapeutic effect. Using these vaccines will make it possible to provide universal protection for humans against rotaviruses.

Description

The invention relates to molecular biology, biotechnology, medicine and is intended for the prevention and treatment of rotavirus infection. A vaccine against rotavirus infection based on a hybrid protein (SEQ ID N0: 1, SEQ ID N0: 2), consisting of the immunogenic epitopes of VP6 and VP8 proteins, connected by a flexible bridge (variants) is proposed. Also proposed is a vaccine against rotavirus infection based on a hybrid protein (SEQ ID NO: 3 - SEQ ID NO: 6), consisting of VP6 and VP8 immunogenic epitopes of proteins and flagellin fragments adjuvant, connected by flexible bridges (variants). Hybrid protein - the active component of vaccine variants - is obtained in prokaryotic and eukaryotic cells using recombinant DNA technology and various purification techniques. The immunological polyvalence of the proposed variants of the rotavirus vaccine of groups A, B and C is shown, and the preventive and therapeutic action is shown. The use of these vaccine options will provide universal protection for humans against rotaviruses.

Technical field

The invention relates to molecular biology, biotechnology, medicine and can be used as a means for the prevention and treatment of rotavirus infection caused by various strains of rotavirus.

Prior art

The term hybrid (chimeric) protein in this text refers to a protein resulting from the expression of a recombinant DNA molecule in which the coding regions of several different genes are connected to each other in the same reading frame.

According to the World Health Organization, rotavirus (the genus Rotavirus, family Rotaviridiae) is one of the leading causes of severe diarrhea, leading to dehydration in young children. Most children become infected before the age of 5 years. Most cases of acute rotavirus gastroenteritis occur in low-income countries, affecting children under the age of 1 year. In most countries with low incomes in Asia and Africa, the epidemiology of rotavirus infection is characterized by episodes of relatively intense virus circulation compared with background transmission throughout the year. However, in temperate countries with high incomes, typical winter seasonality is typical [1].

Rotavirus damages the enterocytes located on the microvilli of the small intestine, which leads to a decrease in absorption and diarrhea. A wide range of clinical manifestations range from transient mild diarrhea to severe diarrhea and vomiting, causing dehydration, electrolyte imbalance, shock and untreated death.

Immunity to rotavirus infection in most cases occurs in early childhood after an illness. Immunity is unstable, so in adults with low levels of antibodies, the disease can recur. Immunity in recovering patients is caused not only by humoral, but also by secretory antibodies.

Registered vaccines for the prevention of rotavirus infection contain live attenuated strains of human and / or animal rotavirus, which multiply in the human intestine. There are two rotavirus vaccines on the international market: monovalent (RV1) and pentavalent (RV5). The RV1 vaccine is derived from the human strain, while the RV5 vaccine contains 5 recombinant viruses derived from the human and bovine rotavirus strains.

The use of vaccines based on recombinant proteins avoids the risks associated with the introduction of the virus into the body, even if it is attenuated. Thus, the selected areas of proteins do not carry the functions of natural proteins of rotaviruses (for example, hemagglutinating activity) and, accordingly, do not cause side effects characteristic of vaccines obtained using rotaviruses. Vaccine preparations obtained using recombinant DNA technology have a higher pharmaceutical purity (do not contain allergens in the form of egg proteins) than virions grown in chicken embryos and do not contain preservatives (formaldehyde, merthiolate). The technology of producing recombinant proteins using bacterial cells makes it possible to obtain, within 2-3 days in fermenters of small volume, which occupy a smaller area and require less technical resources, compared with the cultivation of a live virus, a similar or greater amount of vaccine doses (10-liter fermenter ensures on average, 1,000,000 doses).

The outer capsid protein VP4 is responsible for the adaptation of rotaviruses to in vitro growth, controls their virulence at the organism level, is a viral hemagglutinin and, apparently, ensures the penetration of the virus into the cell. In experiments with neutralizing monoclonal antibodies to VP4, they were shown to impart protective immunity to the body against homologous and some heterologous serotypes of rotavirus.

The VP8 protein is an N-terminal tryptic fragment of the VP4 protein. This protein (VP8) is a highly immunogenic polypeptide and induces an effective homotypic protection against the disease, and the effect was observed in the young of the females grafted with this polypeptide. The VP8 protein contains five neutralizing epitopes.

The use of VP8 protein epitopes to create a vaccine against rotavirus infection will provide protective immunity against homologous and some heterologous rotavirus serotypes.

The polypeptide of the inner capsid VP6 (42 kD) is a major virion protein. The introduction of this protein induces the production of neutralizing secretory antibodies (IgA). Such antibodies are found in large quantities in the milk of lactating females, which ensures the protection of newborn animals [2].

Thus, the child can be protected from rotavirus infection by vaccinating the mother with the proteins VP6, VP8. IgA antibodies are also secreted on epithelial cells, which protects the adult from infection. It is more convenient to vaccinate with a single construct containing the sequences VP6, VP8. Production of a chimeric protein containing not full-length proteins, and their immunogenic epitopes, is more beneficial.

- 1,031404

A known chimeric protein containing a rotavirus subunit protein or its immunogenic part, as well as another protein, an adjuvant is used in conjunction with a chimeric protein [3]. Another protein does not affect the expression and immunogenicity of the rotavirus protein, prevents the formation of complexes by a rotavirus subunit protein and ensures its purification. The rotavirus subunit protein is selected from VP1, VP2, VP3, VP4, VP6, VP7, NSP1, NSP2, NSP3, NSP4 or NSP5.

The invention is known, in which the epitope of a virus of the virus causing epizootic diarrhea of pigs (PEDV) and a protein of rotavirus (VP8 or BVP5) is expressed in a transformant [4]. These polypeptides are functionally linked in a single vector construct. Also claimed is a vaccine composition, which may contain the recombinant protein obtained in the transformant. The purpose of the vaccine is to induce the formation of antibodies neutralizing the indicated viruses and to reduce the degree of their infection in animals. A recombinant vector is created, the vector is introduced into Agrobacterium, followed by transformation of plant cells with these bacteria.

A vector construct for the expression of a target gene in plants is known, the target gene is a gene from the group of GFP, rotavirus proteins VP6, VP2, VP4 and VP7 [5]. It is provided for the transformation of plant cells by bacteria containing the vector structure described.

It is more promising to use several genes encoding immunogenic epitopes of rotavirus proteins in one design and, accordingly, one hybrid protein providing a wider range of activities, respectively, providing a greater degree of protection against the pathogen.

A recombinant carrier protein (VP6) is known, in the sequence of which epitopes of other proteins of other viruses are introduced, with VP6 epitopes preserved on the protein surface [6].

The disadvantages of analogues can be attributed to the limited nature of their actions due to the use of only one rotavirus protein as a component of the hybrid protein. Also the disadvantage of the analogs is the difficulty of obtaining the active protein due to the complexity of its proper spatial folding due to the use of the full-length rotavirus protein as a component of the hybrid protein.

The prototype of the invention (hybrid protein SEQ ID NO: 1, SEQ ID NO: 2) is a recombinant chimeric protein carrying epitopes of VP4 proteins of rotavirus P [4] 223, P [4] 56 and VP7 of rotavirus G1 [142], G4 [208] , P [8] 296 and G2 [87] [7].

The disadvantages of the prototype can be attributed to the limited immune response to rotavirus infection: only one mechanism - the synthesis of neutralizing antibodies that prevent the virus from entering the enterocyte. Such a mechanism provides only the preventive effect of the vaccine.

A recombinant gene is known that contains the nucleotide sequence encoding the first H1-d epitope of the Salmonella structural flagellin gene and at least one epitope of a heterologous organism included in the flagellin sequence in such a way that flagellin is able to bind to flagellin antibody, and at least one epitope of the heterologous organism is placed instead of DNA located between the natural EcoRV sites of the H1-d Salmonella gene [8]. In the present invention, only the first epitope of the H1 (FliC1) Salmonella gene is claimed as part of the chimeric protein, and VP7 protein is indicated in the examples as the second possible structural element. Using both epitopes - FliC1, FliC2 - in a single design will allow you to get a stronger immune response to the introduced immunogen, using only epitopes of the H1 gene connected by flexible bridges, as well as using several immunogenic epitopes of proteins that induce the formation of an immune response by different mechanisms, VP6 and VP8 , will provide comprehensive protection against the virus.

Known recombinant chimeric proteins, among which proteins are claimed, include fragments of VP4, VP7, FIjb: VP4 1-336 :: VP7 :: FIjb [9]. This invention is closest to the proposed (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6) - the prototype. The disadvantages of this design include the difficulty of obtaining an active, soluble protein due to the occurrence of difficulties associated with the acquisition of the wrong tertiary structure of a fusion protein of considerable size.

The claimed invention relates to objects of the same type of the same purpose, providing the same technical result (options).

The technical result from the use of the invention is to simplify the production of an active soluble fusion protein by reducing the size of the protein due to the use of only protein fragments — immunogenic epitopes linked by flexible bridges.

The technical result from the use of the invention also consists in expanding the spectrum of activities of the hybrid protein based on rotavirus proteins, in addition to providing preventive therapeutic action. Neutralizing IgA antibodies, binding to the cell membrane protein of the virus (VP8), stop the penetration of the virus into the enterocytes and induce its excretion from the cells, and when the VP6 capsid protein binds, the intracellular reproduction of the virus stops directly in the enterocyte. In view of this, it is advisable to use as an immunogen, in addition to the VP4 protein fragment, also the protein of the inner capsid (VP6), since it remains on the surface of the viral particle after entering the enterocyte.

The introduction of rotavirus VP6 epitopes into the hybrid protein construct will stop the replication of the virus inside the enterocytes, by producing antibodies to this protein, which will lead to

- 2 031404 therapeutic effect.

The technical result from the use of the group of inventions mainly consists in increasing the valence of the rotavirus vaccine: after vaccination with the vaccines claimed in the invention, a person develops immunity to rotaviruses of groups A – C. After vaccination, the body produces antibodies to epitopes of VP6 and VP8 proteins of various rotaviruses. Formed the ability to produce antibodies in response to ingestion of rotavirus. In the future, when rotavirus A, B, or C is ingested, the immune response will be quickly produced, which will result in the avoidance of infection, or the disease will be mild. Such an approach to vaccination will eliminate the disease.

The technical result from the use of a vaccine containing a hybrid protein, which additionally introduced fragments of flagellin (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6), is to increase its immunogenicity and more rapid response to therapy and hybrid protein vaccination.

The technical result from the use of the FliCl, FliC2 flagellin epitopes as an adjuvant, a non-toxic agent, in the above protein construct consists in enhancing the immune response and a faster response to therapy and vaccination with a hybrid protein. At the moment, flagellin is one of the most promising and well-studied adjuvants of the new generation. Research results show that recombinant proteins introduced with flagellin have enhanced immunogenic and antigenic characteristics. Responses to them are recorded in a shorter time and cause a stronger cellular and humoral immune response [10].

The technical result from the production of only one component - the hybrid protein - instead of the production of many individual components of the vaccine mixture (analogs) is reflected in the cheaper and shortened production time of the vaccine.

Summary of Invention

The proposed invention is a vaccine based on highly purified hybrid proteins (SEQ ID NO: 1, SEQ ID NO: 2), including the immunogenic epitopes of the VP6 protein, the immunogenic epitope of the VP8 protein of rotavirus A, the fragment of the VP6 protein has common and homologous regions with the fragment of the VP6 protein rotavirus C, as well as homologous sites with a fragment of the VP6 protein of rotavirus B, the components are connected by flexible bridges. The group of inventions also includes vaccines, the active component of which (described hybrid proteins) additionally contains the components of flagellin FliC1 and FliC2 as an adjuvant (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6) Components connected by flexible bridges. The presented fragment of the VP6 protein is a consensus sequence to which, in the course of a natural infection, specific antibodies are formed that cross-react with homologous epitopes among different rotavirus strains of groups A, B and C. This fragment contains two immunogenic epitopes. The use of epitopes of several proteins allows to increase the effectiveness of the vaccine. The VP8 protein fragment used contains one highly immunogenic epitope. The use of flexible bridges between epitopes allows you to maintain the correct spatial folding of the protein and, accordingly, ensures the full functioning of each epitope.

Vaccines obtained by a method that includes the creation of a codon-optimized expression for expression in prokaryotic or eukaryotic cells of recombinant DNA encoding a hybrid protein (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6), the introduction of such DNA into a vector construct, ensuring a high level of its expression in prokaryotic cells, or eukaryotes, the introduction of this vector construct into prokaryotic cells, or eukaryotes, the production of a hybrid protein in a specified organism, isolation, purification and mixing with physiological ki acceptable carrier.

Based on the selected amino acid sequences, the nucleotide sequences of the corresponding genes were calculated taking into account the frequency of occurrence of codons in the protein-coding genes of the used prokaryotes or eukaryotes, while the codons were chosen taking into account the decrease in dG of the corresponding mRNA.

To obtain hybrid proteins in accordance with the present invention, standard molecular biology and microbiology techniques known to those skilled in the art can be used. Such methods are fully represented in the scientific literature.

Brief Description of the Drawings

FIG. 1 - the dynamics of the death of mice immunized with a candidate vaccine (SEQ ID NO: 6), intranasal administration, after infection of 10LD / 50 of rotavirus antigenic group A;

FIG. 2 - the dynamics of the body weight of surviving mice immunized with a candidate vaccine (SEQ ID NO: 6), intranasal administration, after infection of 10LD / 50 of rotavirus antigenic group A;

FIG. 3 - the dynamics of the death of mice immunized with a candidate vaccine (SEQ ID NO: 5), intramuscular administration, after infection of 10LD / 50 of rotavirus of the antigenic group B;

FIG. 4 shows the body mass dynamics of surviving mice immunized with a candidate vaccine (SEQ ID NO: 5), intramuscular administration, after infection of 10LD / 50 of rotavirus of the antigenic group B;

- 3 031404 of FIG. 5 - the dynamics of the death of mice immunized with a candidate vaccine (SEQ ID NO: 2), intramuscular administration, after infection of 10LD / 50 of rotavirus antigenic group C;

FIG. 6 - body mass dynamics of surviving mice immunized with a candidate vaccine (SEQ ID NO: 2), intramuscular administration, after infection of 10LD / 50 rotavirus of the antigenic group C. Serum antibody titers (IgA) after twice immunization of mice with a hybrid protein (SEQ ID NO: 1 , SEQ ID NO: 3)

Sorbed antigens (virus) Rotavirus A Rotavirus B Rotavirus C IgA titers in the serum of Balb / c mice, fusion protein (SEQ ID NO .: 1) 65 300 5,200 14,400 IgA titers in the serum of Balb / c mice, Hybrid protein (SEQ 1D NO .: 3) 70,000 5,400 15,000

Bibliography

1. http://www.who.int/immunization/documents/WHO_PP_Rotavirus_2013_summary_RU. pdf-print 10.03.2013

2. Gil MT.de Souza CO, Asensi M, Buesa J., Homotypic protection against subtypes of VP4 capsid protein. Viral Immunol. 2000; 13 (2): 187-200

3. US1,9980106347P 19981030

4. KR20120066559 (A), 20101214

5. K.R20060013000,20040805

6. CN102643335 (A), 20120417

7. CM 102643348 (A), 20120417

8. US 19920837668 19920214-expired

9. CA20102698413 20100406

10. Balaram P, Kien PK, Ismail A. Toll-like mycobacterial and Salmonella infections. Int J Med Microbiol. 2009 Mar; 299 (3): 177-85. doi: 10.1016 / j.ijmm.2008.08.004. Epub 2008 Oct 8. Review.

11. Yamada K., Wang J.C., Osawa H. et al. Efficient large-scale transformation of yeast // Biotechniques .- 1998.- V.24.- P.596-598

12. Bogush V.G. et al., 2009, J. Neuroimmune Pharmacol. v.4, 17-27

13. Komarova, T.B., Skulachev, M.V., Zvereva, AS, Schwartz, Α.Μ., Dorokhov, Yu.L., Atabekov, I.G. (2006). Biochemistry, 71, 1043-1049

The implementation of the present invention

Example 1. Preparation of a highly purified hybrid protein (SEQ ID NO: 1) for a rotavirus vaccine using E. coli bacteria cells.

1.1. Simulation of the hybrid protein.

Planned hybrid polypeptide has 2 domains: VP6, VP8. The following actions were performed to model the protein.

1. Search for immunogenic epitopes of proteins VP6, VP8 using the CPREDS program (http: // ailab. Cs.iastate.edu/bcpreds/predict.html).

2. Building a model of a whole protein to determine the orientation of domains.

3. Building models for each domain (using samples of 3D structures and ab initio).

4. Docking models using the whole protein model.

To obtain the most realistic results in the automatic mode, the I-Tasser algorithm was used to model proteins.

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The simulated fusion protein consists of 175 amino acid residues; its amino acid sequence is represented as SEQ ID NO: 1. Amino acid sequence analysis of this protein using the ProtParam program (http://au.expasy.org/tools/protparam.html) showed that the fusion protein is stable and has a molecular weight of 19.4 kDa, pI 4.41.

1.2. Creating a nucleotide sequence that encodes a hybrid protein.

Translated the amino acid sequence of the hybrid protein, including the VP6, VP8 domains, into the nucleotide (525 bp), optimizing the latter for expression in P. coli cells.

The synthesis of this nucleotide sequence was carried out by lengthening mutually overlapping oligonucleotides according to the described methods (Majumder, 1992). Oligonucleotides were fragments of a hybrid gene with a length of about 70 nucleotides with overlapping regions of a length of about 20 nucleotides. The basic requirements for the primers were that their length should not exceed 60 nucleotides, and the hybridization sites should be at least 20 nucleotides. In addition, at the ends of the oligonucleotides there should not have been long sections with repeated G or C. In some cases, the selection of optimal primers was carried out empirically by shifting the primer relative to the template or changing the length of the primer by 3-6 nucleotides. In total, for the synthesis of a hybrid gene 525 bp in length. 20 primers were used. Synthesized fragments of 300 p. were isolated by gel electrophoresis and cloned into the plasmid vector pGEM-T Easy. Cloning was performed using restriction sites KpnI, SacII, EcoRV, BamHI or by blunt ends. After sequencing, the fragments were amplified, after which they were combined into the nucleotide sequence of the hybrid protein by their fusion by the method of polymerase chain reaction (PCR). After the final synthesis of the hybrid gene by ligating the fragments, the artificial gene was cloned into the pGEM-T vector at the restriction sites KpnI and SacI. The resulting gene was flanked by additional EcoRI restriction sites at the 5'-end and XhoI at the 3'-end. Next, the artificial gene was re-cloned into the pET24a expression vector by restriction sites EcoRI and XhoI.

1.3. Creating a plasmid DNA that encodes a hybrid protein.

The resulting gene was cloned into the pET24a plasmid for subsequent expression. For this, a ligation of the gene and the pET24a vector was performed using the appropriate buffer and ligase at 20 ° C for 2 h.

The mixture was heated at 95 ° C for 10 min and purified from salts by dialysis on nitrocellulose filters with a pore diameter of 0.025 μm (Millipore, USA). Dialysis was performed against a solution containing 0.5 mM EDTA in 10% glycerol for 10 minutes.

1.4. Creating a strain of E.coli for amplification of plasmid DNA containing the hybrid gene.

The resulting plasmid was transformed into E cells. This strain DH10B / R (Gibko BRL, USA) with the F-mcrA A genotype (mrr-hsdRMS-mcrBC) p80dlacZAM 15 AlacX74 deoR recA1 endA1 araD139 A (ara, leu) 769 galU galXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXAXAXAXAAxA3 electroporation.

After transformation, cells were incubated in SOC medium (2% bacto-trypton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) for 40 min at 37 ° C .

Using E. coli cell screening for the presence of plasmids on selective medium containing LBarap, 100 μg / ml ampicillin, E. coli cells were selected — E. coli strain for amplifying plasmid DNA containing the hybrid gene.

Plasmid DNA was isolated from the grown clones using the Wizard Minipreps DNA Purification System kit (Promega, USA).

Purified plasmid DNA was verified by restriction analysis and sequencing. In the course of the work, clones were selected that contain DNA fragments of the required size in the composition of the plasmid, from which such plasmids were isolated to further induce the expression of the gene.

1.5. Creating a strain of E.coli - producer of the hybrid protein.

For the expression of the gene encoding the fusion protein, E. coli cells of the BL21 Star (DE3) strain (Invitrogen, USA) were used, with the F-ompT genotype hsdSB (rB-mB-) gal dcm rne131 (DE3), containing lysogen in the XDe3 genome mutation rne131. The mutated me (rne131) gene encodes a truncated form of RNase E, which reduces the intracellular destruction of mRNA, leading to an increase in its enzymatic stability. lon and ompT mutations in protease genes allow for the production of non-proteolyzed recombinant proteins in large quantities.

ExAI cells of the strain BL 21 with the genotype F- ompT hsdSB (rB-mB-) gal dcm rne131 (DE3) were prepared as follows. Incubated the cells at 37 ° C overnight in 5 ml of L-broth containing 1% tryptone, 1% yeast extract and 1% sodium chloride. The culture was diluted with fresh L-broth 50-100 times and grown on a rocking chair at 37 ° C to an optical density of 0.2-0.3 at a wavelength of 590 nm. Upon reaching an optical density of more than 0.3, the culture was diluted with fresh L-broth to an optical density of 0.1 and grown for 30 minutes. Transferred 100 ml of the culture to a sterile centrifuge tube and precipitate the cells at 4 ° C for 5000xg for 10 minutes. The supernatant was discarded, the cells were resuspended in deionized water in the original volume, followed by centrifugation. About

- 5 031404 cleaning procedure was repeated three times. After washing, the cell pellet was resuspended in a small volume of deionized water and centrifuged for 30 s. at 5000 rpm on a microcentrifuge.

Transformation of competent cells was carried out by electroporation. For this, 1 μl of plasmid DNA was added to 12 μl of competent cells, mixed, and electroporated on a high-voltage HVI-1 impulse generator (St. Petersburg State Technical University, St. Petersburg) in sterile cells with a 4 ms electric pulse.

After transformation, cells were incubated in SOC medium (2% bacto-trypton, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) for 40 min at 37 ° C. 10-100 μl of cell suspension were sown on selective LB-medium (Gibko BRL, USA) containing ampicillin (100 μg / ml) for the selection of clones containing plasmids (producer strains).

The plasmid obtained after transformation of competent cells of B. coli strains provided a high level of the biosynthesis of the recombinant protein encoded in it.

1.6. Obtaining a hybrid protein in E. coli cells by induction of protein synthesis with 0.2% lactose by the method of Studier.

For the cultivation of the obtained producer strains, standard agarized LB medium was used containing ampicillin at a concentration of 100 μg / ml and glucose at a concentration of 1% to block non-specific expression.

Induction of expression was performed when the cell culture reached an optical density of 0.6-0.8 optical units at a wavelength of 600 nm.

0.2% lactose was used as an inducer (Studier, 2005).

For autoinduction of expression according to the Studier method (Studier, 2005), PYP-5052 medium was used consisting of 1% peptone (Gibco, USA), 0.5% yeast extract (Gibco, USA), 50 mM Na ₂ HPO ₄ , 50 mM K2HPO4 , 25 mM (NH4) 2SO4, 2 mM MgSO4, 0.5% glycerol, 0.05% glucose and 0.2% lactose.

On Wednesday, PYP-5052, containing ampicillin at a concentration of 100 μg / ml, was inoculated with a single colony of the producer strain. Fermentation was carried out at 37 ° C in a thermostatically controlled rotary type shaker at 250 rpm for 20 hours until there was no significant change in OD ₆₀₀ for 1 hour. An aliquot of cells was taken for analysis of the expression of the gene encoding the vaccine protein by electrophoresis in PAAG precipitated by centrifugation at 9000xg.

Protein was isolated from E. coli cells by cell lysis. Cells were resuspended in lysis buffer containing 20 mM Tris-HCl pH 7.5, 5 mM EDTA and 1 mM phenoxymethylsulfonyl fluoride, based on 1 g of cells 5-7 ml of buffer. The cell suspension was treated with ultrasound 7 times for 30 s with an interval of 30 s (the ultrasound frequency is 22 kHz). The lysate was centrifuged for 10 min at 4 ° C, 5000xg. The supernatant was decanted, a solution of 1 M urea at the rate of 10 ml per 1 g of cells was added to the precipitate, mixed vigorously. Repeated centrifugation. The supernatant was decanted, the pellet was resuspended in a solution of 2 M urea of the same volume. Repeated centrifugation. The supernatant was discarded.

The obtained preparation contained, according to SDS-PAGE (PolyAcrylamide Gel Electrophoresis with Sodium dodecyl sulfate), about 98% of the hybrid protein at a concentration of 1 mg / ml.

The conditions of isolation and purification were selected experimentally and can vary in the values known to the average expert in this field.

1.7. Obtaining a purified preparation of the hybrid protein.

MgCl2 was added to the hybrid protein solution to a concentration of 6 mM and purification of the hybrid protein was performed using immobilized metal affinity chromatography (IMAX) using Ni-NTU sepharose sorbent. Binding to this sorbent occurs at the expense of 6 histidine residues present at the N-terminus of the obtained recombinant protein. The protein solution was filtered through a PVDF filter with a pore diameter of 0.22 μm, to the resulting filtrate was added sorbent Ni-NTU Sepharose, equilibrated with refolding buffer, on the basis that 1 ml of sorbent binds no more than 40 mg of protein, and the binding was carried out with constant stirring in for 2 hours. The sorbent was precipitated by centrifugation and applied in a minimum volume onto a gravity chromatographic column containing 2 ml of sorbent.

The elution was performed by a step-like imidazole gradient from 20 to 300 in increments of 50% from the previous one, with each step having a volume of elution solution equal to 5-20 column volumes. The yield and purity of the protein was controlled by the method of disk electrophoresis and measurement of protein concentration by the method of Bradford.

To determine the content of endotoxins, a standard stock solution of endotoxin with a concentration of 4000 EU per ml was prepared, the resulting solution was stable when stored at 4 ° C for at least 2 weeks. A series of dilutions of a stock solution of endotoxin in increments of 2 times was prepared using endotoxin-free water in sterile polystyrene tubes sealed with endotoxin-free parafilm. A series of dilutions of the sample of substance was prepared with a concentration step of 5, and after preliminary determination of the endotoxin content, with a concentration step of 2.

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A sample, water or endotoxin in 100 μl was added to the bottom of each eppendorf, after which 100 μl of LAL reagent lysate was added and incubated for 1 h at 37 ° C in a water bath. Results were evaluated by the presence or absence of a dense thrombus at the bottom of the tube by inverting the tube.

It was shown that in the test sample there are no factors that inhibit the formation of thrombus. A gel clot formed at 1/8 dilution of the sample, i.e. when the sensitivity of the method is 0.03 EU / ml, the sample with a concentration of 50 μg / ml contains less than 100EU.

The conformation of the recombinant fusion protein during its synthesis in E. coli cells was determined by the method of electrophoresis disk electrophoresis of E. coli cells destroyed using ultrasound after the induction of expression, and the sediment and the supernatant formed after the precipitation of cell debris were analyzed. As a result of the densitometric analysis, it was shown that the recombinant protein synthesized in the E.coli cells of the producer strains with the induction of expression of 0.2% lactose is 100% in the supernatant, i.e. soluble fraction.

Cultivation of bacteria at different temperatures: 20, 30, and 37 ° C did not cause a change in protein solubility.

After carrying out a disk electrophoresis in PAAG of lysates of E. coli cells after autoinduction of expression with 0.2% lactose, the expression level of the target protein in E. coli cells was analyzed by densitometry. As a result of a densitometric analysis, it was shown that in E. coli cells the hybrid protein accumulates in the amount of 45% of the total cellular protein.

The obtained expression level did not change in the producer strain for 6 passages, which indicates a stable expression pattern of the analyzed gene.

1.8. Preparation of recombinant fusion protein using culturing E. coli cells with continuous addition of nutrient substrates, preferably glucose and yeast extract.

When cultivating the Escherichia coli strain, a batch culture method was used to produce a fusion protein. This method is used to prevent the negative effects of the substrate limit, while the substrate or other necessary components are added either continuously or by a signal from any sensor. To optimize the yield of products released into the medium, it is important to strengthen the biosynthetic ability of bacterial cells, and the feeding method of cultivation allows you to extend the second phase of growth and increase the yield of extracellular metabolites. This method can be used in the case of a potentially toxic substrate (recombinant proteins are toxic to E. coli cells), since its concentration in the medium will be maintained at a low level.

Limiting the rate of absorption of the substrate by the speed of its delivery is a way to overcome the catabolite repression of product formation.

Culture with water was the most effective way to achieve high cell density and high productivity.

The strain of E.coli was sterile subcultured on beveled shoals of EDTA-containing agar and kept in a thermostat for 5 days. To obtain the inoculum, cultures were washed from the jambs, seeded in a liquid medium with EDTA, and cultured for 3-4 days. After the inoculum in the amount of 10 ml was transferred into a sterile 750-ml flask with 200 ml of sterile liquid medium and cultured for 10 days on a rocking chair at 150-200 rpm at a temperature of 28-30 ° C.

Solid nutrient medium was used to obtain a fresh culture of the strain.

Liquid nutrient medium was used to obtain inoculum and for periodic cultivation.

Preparation of liquid nutrient medium.

1) MgSO4-7H2O 10 ml / l.

2) edO ^ BUT 20 ml / l.

3) KH ₂ PO ₄ + NaH ₂ PO ₄ -12H ₂ O 10 ml / l.

4) EDTA 10 ml / l.

5) Trace elements 1 ml / l (adjusted to pH 4.2) + 5.6 g / l EDTA:

FeCl ₂ -4H ₂ O 1.5 g / l;

Н3ВО3 0.06 g / l;

MnCh ^^ 0.1 g / l;

CaCl ₂ -6H ₂ 0 0.12 g / l;

ZnC12 0.07 g / l;

NiCl ₂ -6 H ₂ O 0.025 g / l;

CuCl ₂ -2 H ₂ O 0.015 g / l;

Na2MoCl4 0.025 g / l.

6) Vitamins:

pyridoxine 20 mg;

- 7 031404 thiamine 10 mg;

Riboflavin 10 mg;

nicotinic acid 10 mg;

P - aminobenzoic acid 10 mg;

lipoic acid 10 mg;

nicotinamide 10 mg;

Vitamin B12 10 mg;

Biotin 4 mg;

folic acid 4 mg.

All components were dissolved in 200 ml of water, sterilized at 0.5 atm. 30 minutes and added to the liquid nutrient medium in the amount of 1 ml / l.

The components of the nutrient media were weighed on technical and analytical electronic scales and dissolved in distilled water. Experience in the preparation of nutrient media showed that it is convenient to prepare it from pre-sterilized concentrated solutions.

Solid nutrient medium was prepared as a liquid, with the addition of 3% agar.

The optical density (OD ₆₀₀ ) of E.coli cell cultures was 9 O.E. Cells were pelleted by centrifugation at 13000xg for 6 min at 10 ° C. The cell pellet was weighed, it was 1.9 g.

The cells were resuspended in 30 ml of buffer (50 mM Tris-HCl, pH, 50 mM EDTA, 20 mM L-cysteine, pH8.6) and destroyed using ultrasound (sounding time - 10 min, pulse time - 30 s, pause time between pulses - 30 s, amplitude - 70%). After cell disruption, inclusion bodies were precipitated using centrifugation at 30000xg for 20 minutes at 10 ° C, the weight of the crude inclusion bodies from the cells was 1.5 g. The precipitated inclusion bodies were washed using a sequential change of several buffers as follows.

1. The precipitate Taurus inclusion resuspendable in 10 ml (last wash 15 ml) of buffer for washing.

2. The resuspended inclusion bodies were stirred on a horizontal shaker for 1 hour at room temperature.

3. The inclusion bodies were precipitated by centrifugation at 30000xg for 20 minutes at a temperature of 10 ° C.

After five washes, the wet weight of the inclusion bodies was 0.9 g.

To solubilize the protein from the washed inclusion bodies, 18 ml of solution was used (9 M urea, 2 mM EDTA, 50 mM Tris-HCl, pH8.6). The solubilized inclusion bodies were centrifuged at 30000xg for 20 minutes at a temperature of 10 ° C. The resulting supernatant was transferred to new falcons and used for refolding.

Refolding was performed by the method of 10-fold dilution of solubilized inclusion bodies in the refolding buffer at a temperature of 4 ° C dropwise. MgCl2 up to 6 mM was added to the solution of the refolded protein and immobilized metal affinity chromatography (IMAX) was used for protein purification using Ni-NTU Sepharose sorbent. The protein solution was filtered through a PVDF filter with a pore diameter of 0.22 μm, and a Ni-NTU Sepharose sorbent equilibrated with refolding buffer was added to the obtained filtrate, on the basis that 1 ml of sorbent bound no more than 40 mg of protein, after which binding was carried out with constant stirring for 2 hours. The sorbent was precipitated by centrifugation and applied in a minimal volume onto a gravity chromatographic column containing 2 ml of sorbent. Elution was performed by a step gradient of imidazole (20, 40, 100, 150, 200 and 300 mM), with each step having a volume of eluting solution equal to 10 column volumes.

Using this purification method, 43 mg of the hybrid protein was obtained with a purity of 98%.

1.9. Preparation of a fusion protein, purified before dissolution, by removal of soluble cellular components, including DNA, RNA, proteins, lipopolysaccharides, by washing with a buffer solution containing the detergent guanidine hydrochloride.

After the fermentation and destruction of E. coli cells and washing of inclusion bodies (item 1.8.), Protein solubilization was performed with 18 ml of a solution (8 M GuHCl, 2 mM EDTA, 50 mM Tris-HCl, pH 8.6) containing guanidine hydrochloride. The solubilized inclusion bodies were centrifuged at 30000xg for 20 minutes at a temperature of 10 ° C. The resulting supernatant was transferred to new falcons and used for refolding.

Next, the recombinant protein was refolded and purified (see Example 8). As a result of the purification, 41 mg of the hybrid protein was obtained with a purity of 98%.

Example 2. Preparation of a highly purified fusion protein (SEQ ID NO: 3) for a rotavirus vaccine using E. coli cells.

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2.1. Simulation of the hybrid protein.

A planned hybrid polypeptide is a complex multi-domain protein (4 domains: FliCl, FliC2, VP6, VP8). To simulate multi-domain proteins, the following actions were performed.

1. Definition of domain boundaries.

2. Building a model of a whole protein to determine the orientation of domains.

3. Building models for each domain (using samples of 3D structures and ab initio).

4. Docking models using the whole protein model.

In the planned hybrid polypeptide, two domains had samples, and two needed ab initio modeling; in addition, ab initio modeling required the formation of flexible bridges between the domains.

To obtain the most realistic results, the I-Tasser algorithm, recognized as the best in the last three CASP (Critical Assessment of protein Structure Prediction) - protein modeling competitions, was used in automatic mode. This analysis was carried out for four days. However, even using this powerful algorithm, obtaining adequate data for a multi-domain protein with the need for ah initio modeling of domains and their boundaries is not completely reliable (75%).

To obtain more accurate data, we divided the proteins into used domains, carried out their modeling using I-Tasser, and then carried out their docking.

The simulated hybrid protein consists of 458 amino acid residues; its amino acid sequence is represented as SEQ ID NO: 3. Amino acid sequence analysis of this protein using the ProtParam program (http://au.expasy.org/tools/protparam.html) showed that the fusion protein is stable and has a molecular weight of 48.6 kDa, pI 4.6.

2.2. A nucleotide sequence encoding a hybrid protein was created according to Clause 1.2 of Example 1, a plasmid DNA encoding a hybrid protein was created according to Clause 1.3 of Example 1, an E. coli strain was created to amplify plasmid DNA containing the hybrid gene according to Clause 1.4. Example 1, created E.coli strain producing hybrid protein, according to 1.5. of example 1, the obtained hybrid protein in E. coli cells by induction of protein synthesis with 0.2% lactose according to the method of Studier, according to p.1.6 of example 1, the purified preparation of the hybrid protein was obtained, according to p.1.7. Example 1. The protein yield was 1 g with 1 l of medium.

Example 3. Obtaining highly purified hybrid protein (SEQ ID NO: 2), for a vaccine against rotavirus infection, using the cells of the yeast S.cerevisiae.

A hybrid protein was simulated in accordance with Clause 1.1 of Example 1, and the nucleotide sequence encoding the hybrid protein was generated in accordance with Clause 1.2. of example 1 (the nucleotide sequence is optimized for gene expression in yeast), a plasmid DNA encoding a fusion protein was created according to section 1.3. Example 1 (used vector pYES2 / NT, Invitrogen, USA).

3.1. Creating a strain of Saccharomyces cerevisiae - producer of hybrid protein.

The yeast was cultivated in YPD medium until the middle of the logarithmic growth phase was reached, washed with 10 mM TE buffer. Cells contained in 100 ml of medium were resuspended in 5 ml of 10 mM Tris HCl buffer (pH 7.5) containing 40.5% PEG-4000, 0.1 M lithium acetate. 1 μg of plasmid DNA was added to 0.5 ml of the cell suspension, mixed and incubated overnight at room temperature. Next, 50 μl of the cell suspension were sown on selective medium for the selection of transformants [11].

3.2. Production of the hybrid protein in the resulting Saccharomyces cerevisiae strain.

The resulting producer strain was grown in YPD medium on a rotary shaker at a speed of 250 rpm at a temperature of 28 ° C for 20-24 hours. In a 5-liter Biostat fermenter containing 2000 ml of YPD medium, 80 ml of seed were injected. Fermentation was carried out at a temperature of 28 ° C, aeration of 1 l / min and a stirring speed of 1000 rpm. 24 hours after seeding, the fermenter fed the culture medium with 50% glucose solution at a rate of 2 ml / h and set the pH of the culture at pH 6.8 ± 0.1, using 10% sulfuric acid and 10% NaOH solutions for adjustment. The average total fermentation time is 72 hours. According to electrophoretic analysis, production of the hybrid protein under these conditions was at least 300 mg / l of culture liquid.

3.3. Isolation and purification of the recombinant fusion protein from the water-insoluble fraction of Saccharomyces cerevisiae cells.

The protein was isolated and purified from the water-insoluble fraction of the producer strain [12]. When increasing the biomass of S.cerevisiae in a 5-liter fermenter on YPD medium without feed in the presence of 2% glucose in the starting medium with one fermentation, an average of 700-900 g of wet cell biomass was obtained. 1.5 kg of the washed wet biomass was resuspended in the buffer for destruction, the cells were destroyed with the help of glass beads in a flow-mill for 1.5 h, the resulting suspension was centrifuged and the precipitate was collected. Extraction of the target protein from the precipitate was carried out using a solution of 10% lithium chloride in 90% formic acid for 16

- 9 031404 h followed by centrifugation. The pellet was discarded, and the supernatant was subjected to ultrafiltration, followed by diafiltration through an M15 membrane to convert to 10 mM sodium acetate, pH 4.0 and remove host cell proteins with a molecular weight below 15 kDa.

The protein was deglycosylated using endo-3-N-acetylglucosaminidase H in 50 mM sodium citrate buffer (pH 5.5), at 37 ° C for 18 hours in the presence of 0.01% SDS.

The final purification was performed using ion-exchange chromatography on a HiPrep 16/10 SP FF cation-exchange column (GE Healthcare) in the FPLS system. After passing the filtrate through the column and then washing the column with 10 mM Na-phosphate buffer, pH 7.0 and then 10 mM sodium acetate buffer, pH 4.0, the protein was eluted from the column with 10% NaCl solution in the same buffer and identified electrophoretic in 10% PAG-DDS-Na, the fractions with the target protein were combined, dialyzed against deionized water, frozen at -70 ° C, and lyophilized. The lyophilized preparation is a white substance similar to cotton wool.

The protein yield was 10 g with 1 l of nutrient medium.

Example 4. Obtaining highly purified hybrid protein (SEQ ID NO: 4), for a rotavirus vaccine, using Pichia pastoris yeast cells.

The hybrid protein was simulated in accordance with paragraph 1.1 of Example 3, a nucleotide sequence encoding a hybrid protein was created in accordance with Clause 1.2 of Example 1 (the nucleotide sequence is optimized for gene expression in yeast), a plasmid DNA encoding a hybrid protein was created according to paragraph 1.3 of Example 1 (used vector pHIL-S1, to obtain secreted protein).

4.1. Preparing cells for transformation.

Spent the cultivation and freezing of the strain of cells of Pichia pastoris SMD1163, defective in several proteases of yeast, which ensures the stability of the secreted protein. Cells were sown under sterile conditions on agar in YPD medium (1% yeast extract, 2% peptone, 2% glucose, 1 mM dithiothreitol), cultured at 30 ° C, then seeded in suspension and cultured overnight. The cell doubling time in the YPD medium of all clones was 2 h. Part of the cells were resuspended in YPD medium supplemented with 15% glycerol and frozen at -86 ° C. To obtain competent cells, cell colonies were preliminarily grown in an agar plate in YPD medium at 30 ° C for 2 days. Then the contents of one colony were grown in 10 ml of YPD medium at 30 ° C overnight. The suspension was diluted in YPD to a density of OD600 0.2 and a final volume of 10 ml and culture was grown to OD (5 ₀₀ 0-8 for 4 h. The cell suspension was centrifuged for 5 min at 500xg, the supernatant was poured, resuspended in 10 ml of solution I below the transformation kit, re-centrifuged and resuspended the pellet in solution I, after which the cells were competent to transform. 50-200 μl aliquots of the competent cells were poured into 1.5 ml sterile tubes that were stored at -90 ° C until use .

4.2. Transformation of competent cells.

For transformation used EasyComp Transformation Kit, part of the Pichia Easy Select Kit (Invitrogen).

3 μg of the DNA construct, 1 ml of solution II from the kit were added to 50 μl of competent cells, mixed by shaking, incubated for 1 h at 30 ° C, stirring the solution every 15 minutes. Then the solution was incubated for 10 min at 42 ° C, divided into 2 microtubes of 525 μl, 1 ml of YPD medium was added to each, and incubated for 60 min at 30 ° C to acquire zeocin resistance cells. After that, the cells were centrifuged for 5 min at 3000xg, resuspended in solution III, adding 500 μl to each tube, poured the suspension from 2 tubes into one, centrifuged again, and resuspended the pellet in 150 ml of solution III. The resulting cell suspension was scattered into a sterile dish (80 mm) on an agar gel prepared on YPD medium with the addition of 1 M sorbitol and the antibiotic zeocin at a final concentration of 100 μg / ml. After 3 days, several dozen colonies were obtained per cup. Cells from the colonies were transferred onto a MMD (minimal medium dextrose) agar plate and the plate was cultured for 2 days at 30 ° C. After this, the culture dish was used to grow the cells in suspension and their subsequent testing.

4.3. Control of expression of the recombinant protein in the resulting strains. Control of expression of the target gene was carried out by electrophoretic separation of proteins in a polyacrylamide gel (SDS-PAGE). For this, cells from colonies grown on selective medium were transferred to flasks and cultured in 5 ml of MGY medium on a rocking chair (250 rpm) for 1 day to OD ₆₀₀ = 5. After that, the cells were precipitated by centrifugation at 3000 xg for 10 minutes. Bio-Rad equipment and ICN reagents were used for electrophoresis. The supernatant and intracellular proteins (cell lysate) were mixed with the application buffer and 10 μl were added to the wells of the concentrating gel. Separation was carried out at a voltage of 150 V for 1 h. Then the gel was stained with the aid of the reagent PAGE blue 83, BDH. Strains were selected for which the presence of bands corresponding to the target protein in molecular weight was detected on electrophoregrams. The level of protein synthesis was determined by comparing the intensity of staining of the recombinant protein band with the band of the corresponding protein, the molecular standard

- 10 031404 mass. As a result, according to the data obtained, the Pichia pastoris 3 strain was selected from 6 producer strains, whose cells synthesize 400 mg of protein per liter of yeast culture.

4.4. Purification of chimeric protein.

After the cells were precipitated, the nutrient medium was filtered (pore diameter 45 μm), then Tris-HCl pH 6.0 was added to a final concentration of 20 mM. The culture medium containing the fusion protein was concentrated 5-10 times using Millipore concentrators for proteins with a molecular weight of more than 10 kDa.

After concentration, the chimeric protein preparation was heated in a water bath to a boil (t = 100 ° C) and boiled for 2 minutes, then centrifuged at 4 ° C, 15000xg for 15 minutes.

Conducted ion exchange chromatography on KM-sepharose. The KM-Separate column was equilibrated with buffer containing 20 mM Tris-HCl pH 6.0. The chimeric protein preparation was applied at a rate of 60 ml / hour. The column was washed with 20 mM Tris-HCl pH 6.0; 20 mM Tris-HCl pH 6.0, 200 mM NaCl. Elution was performed with 20 mM Tris-HCl pH 6.0, 1 M NaCl and 1 ml fractions were collected.

Affinity chromatography was performed on a His-bind® resin column. The column with the carrier was washed with buffer containing 50 mM phosphate pH 8? 0, 500 mM NaCl. The preparation of the chimeric protein obtained after concentration or ion-exchange chromatography was diluted 2 times, phosphate pH 8? 0 was added to a concentration of 50 mM and applied to the column. After washing the column with the deposition buffer, the ballast proteins were removed by washing with a solution of 20 mM imidazole in the same buffer. The protein was eluted with a solution containing 200 mM imidazole.

As a result, a hybrid protein preparation was obtained with a purity of more than 95%.

One of the advantages of the proposed method for the production of recombinant protein is the high technological effectiveness of the protein purification process, due to simple isolation procedures, the relatively low cost of the resins used and the high yield of active protein as a result of purification.

Summarizing the above, we can conclude that the resulting strain of the yeast Pichm pastoris 3 synthesizes chimeric protein in an amount sufficient to isolate it on an industrial scale. As a result, the resulting highly purified chimeric protein can be used as an active component of a vaccine against rotavirus infection.

The protein yield was 24 g with 1 l of medium.

Example 5. Obtaining highly purified hybrid protein (SEQ ID NO: 5) for a rotavirus vaccine using mammalian cells - CHO.

Made a simulation of the hybrid protein according to section 1.1. Example 3, created a nucleotide sequence encoding a hybrid protein according to claim 1.2. of example 1 (the nucleotide sequence is optimized for the expression of the gene in mammalian cells), plasmid DNA encoding a fusion protein was created according to claim 1.3. Example 1 (used vector pSELECT-CGFP-zeo, InvivoGen, USA).

Analysis of the expression of the target gene was performed on a Chinese hamster ovary cell culture (CHO-K1, ATCC CCL 61), as the most widely used in biotechnological practice for producing target proteins.

5.1. Cultivation of cell line CHO-K1.

The CHO-K1 cell line was cultured on DMEM medium containing 10% inactivated fetal calf serum, 2 mM L-glutamine, 35 mg / l L-proline, as well as commercial antibiotic Mycokill-AB (PAA Laboratories) at a working concentration. Cell resection was performed 1 time per 5 days at a cell concentration of 10 ³ cells per 1 cm ² , with a dilution of 1:20. Cells were cultured at 37 ° C, in an atmosphere of 5% CO ₂ and high humidity.

5.2. Transfection of cells.

This cell culture was transfected with the resulting plasmid encoding a fusion protein.

Cells in the amount of 70-80% of the monolayer (8-10 ⁴ cells per 1 cm ² ) were washed with culture medium containing no serum. A transfection mixture was prepared: 3-4 μg of linearized plasmid DNA was mixed with 375 μl of serum-free culture medium. In a separate tube with the same amount of the same medium, the commercial transfection reagent Lipofectamine 2000 was mixed, the amount of which was determined by the ratio of 3 μl of reagent to 1 μg of plasmid DNA. After that, the resulting solutions were combined and incubated at room temperature for 30-40 minutes.

The resulting transfection mixture was layered on the washed cells. After 4-6 hours after the start of the transfection, the transfection mixture was replaced with DMEM culture medium with serum.

The expression level of the target gene was assessed by fluorescence intensity on the second day after transfection (36-48 h) using flow cytofluorimetry. Untransfected CHO-KT cells were used as a negative control.

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5.3. Cytofluorimetric analysis.

For cytofluorimetric analysis, the cells were washed with phosphate-saline buffer, trypsinized, and removed from Petri dishes. Then it was washed twice from trypsin and carefully resuspended in phosphate-buffered saline. The resulting suspension (10 ⁶ cells in 1 ml of buffer) was transferred to 5 ml round-bottomed tubes.

The voltage of the flow cytofluorometer channels was selected so that the autofluorescence of untransfected cells was not taken into account during the measurements. To do this, the voltage in the FL1 channels (in which the eGFP fluorescence is detected) and FL2 (which was used as a control empty channel) was changed so that during the measurement the vast majority of cells not containing the eGFP gene were in the region not exceeding 10 logarithmic scale deferred for channels FL1 and FL2.

After calibration, all samples were measured at constant voltages on channels FL1 and FL2. Thus, if eGFP fluorescence was detected in the cells, the appearance of cells in the region exceeding 10 was recorded on a logarithmic scale on the FL1 channel, and all quantitative values of the gene expression level were measured in this region.

5.4. Maintaining pools of transfected cells.

After transfection, the resulting pools were cultured according to the protocol described above (5.1.).

In order to get rid of cells that do not contain the transgene, selection of the obtained pools of transfected cells was performed using a commercial antibiotic Zeocin (InvivoGen), administered at a concentration of 800 μg / ml. Since, as a result of suppressing the transcriptional activity of the transgene, the production of the antibiotic resistance gene product decreases with time, after 75 days of cultivation, the concentration of the antibiotic was gradually reduced to 200 μg / ml.

5.5. Obtaining and analysis of stable cell lines.

Analysis of heterogeneous cell pools can lead to abnormalities associated with the fact that the transgene during lipofection multicopyingly integrates into different regions of the genome, as a result of which cells may have different viability, speed of division, resistance to the antibiotic and other characteristics important for efficient transgene expression.

Therefore, in practice, recombinant proteins are produced by selecting stable cell lines obtained from a single transfected cell and, thus, characterized by a homogeneous genotype, which eliminates the influence of the above factors and selects the most effective clone.

Individual cell clones were obtained from total cell populations by the method of limiting dilutions after 50 and 75 days of cultivation.

5.6. Getting individual clones.

Cultivated pools were removed from the plates and diluted in 10 ml of culture medium. After this, the cell concentration was calculated using the Gariaev chamber and additional dilutions were made so that the cell concentration was 2-3 cells per 1 ml of medium. The resulting suspension was transferred to 24-well plates 1 ml per well.

After 2 weeks of cultivation on DMEM medium containing antibiotic Zeocin at a concentration of 800 μg / μl, a cytofluorometric analysis of the surviving clones was performed. Further maintenance of the obtained lines and analysis of the expression level of EGFP was performed according to the procedure described for pools of transfected cells.

5.7. The results of the analysis of the temporal expression profile of the EGFP gene in individual cell clones.

The main quantitative indicator of the expression activity of the chimeric gene in this case was the median of the distribution of cells according to fluorescence intensities.

A sufficiently high level of expression of EGFP was observed in two clones with the introduced construct.

After two and a half months of cultivation, the expression activity of clones containing the construct with the gene of interest averaged about 70% of the original.

To obtain a highly purified chimeric protein, CHO cells were transformed with a different plasmid providing the synthesis of the target protein; the protein produced was purified by anion-exchange chromatography on Q-sepharose at pH 7.0. The yield of the target protein was 10 mg / L.

The application of the method for producing a hybrid protein in mammalian cells is possible using also other mammalian cells, for example HEK293-EBNA1.

Example 6. Obtaining highly purified hybrid protein (SEQ ID NO: 6) for a rotavirus vaccine using plants.

Made a simulation of the hybrid protein according to section 1.1. Example 3 created a nucleotide sequence encoding a hybrid protein according to claim 1.2. of example 1 (the nucleotide sequence is optimized for the expression of the gene in plant cells) created plasmid DNA encoding a hybrid protein, according to claim 1.3. example 1.

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To assess the expression of the hybrid protein in plants of Nicotiana benthamiana, the vector was introduced into the strain Agrobaterium tumefaciens GV3101, and recombinant agrobacteria were used to infiltrate N. benthamiana leaves. Agrobacteria containing recombinant binary vectors were grown for 12 hours on a shaker at 30 ° C. Cells (1.5 ml) were precipitated by centrifugation (4000xg, 5 min), the precipitate was resuspended in 1.5 ml of buffer containing 10 mM MES (pH 5.5) and 10 mM MgCl ₂ , the optical density was adjusted to OD ₆₀₀ = 0.2. The leaves of N. benthamiana plants were injected with a suspension of agrobacteria using a syringe without a needle. Infiltrated leaves were left on growing plants.

After N. benthamiana leaves infiltrate with agrobacteria with a virus-vector, most of the leaf cells in the infected area become infected, DNA is transferred to the nucleus, and transcription of a copy of viral RNA occurs. At a subsequent stage, the viral vector replicates and the product is synthesized at a high level [13]. The maximum synthesis of the product is achieved at 6-10 days after infiltration.

The presence of the target protein in the leaves of the producing plants was recorded by electrophoresis in PAGE. At 6–10 days after infection, the leaf fragment was triturated in 0.4 M sucrose buffer, 50 mM Tris pH 8.0, 10 mM KCl, 5 mM MgCl ₂ , 10% glycerol, 10 mM P-mercaptotanol. The obtained extract was centrifuged for 10 min at 14000xg, the supernatant (soluble fraction) and the dissolved precipitate (membrane fraction) were analyzed using polyacrylamide electrophoresis (PAAG) gradient (8-20%) Laemley gel and Coomassie R-250 staining.

Based on the obtained results, in plants infiltrated by a virus-vector in the membrane fraction of cells, a product was found that corresponds in molecular weight to 48.6 kDa. In the control uninfiltrated plant this protein was absent. The product yield was about 6% of the fraction of insoluble proteins.

Based on the results obtained, the claimed hybrid proteins can be obtained using both prokaryotic and eukaryotic cellular systems. A highly purified protein preparation can be obtained using various types of protein purification.

Example 7. Determination of the immunogenicity of the preparation of a hybrid protein (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4).

The immunogenicity of the preparations of the obtained proteins was evaluated. Immunogenicity is the ability of an antigen to initiate the immune system to form effectors that neutralize antigenic foreignness.

Immunization of Balb / c mice (females) was performed intraperitoneally, 20 μg of fusion protein was injected. Immunization was performed twice with an interval of 2 weeks. Animals were divided into experimental and control groups of 7-8 mice in each.

Blood samples were obtained from 7-8 mice of each group 2 weeks after the second immunization from the tail vein. To obtain serum, the blood was incubated for 30 min at 37 ° C. After the formation of blood clots, the samples were placed on the ice surface and cooled for 1 hour, followed by centrifugation for 15 min at 400xg. Blood serum from mice of each group was pooled and frozen at minus 20 ° C.

Antibody titers in the sera of immunized mice were determined using enzyme immunoassay (ELISA). ELISA was performed by the conventional method. Used 96-well plates (Greiner, Germany).

The fusion protein was denatured as follows. Tween-20 detergent was added to the protein sample to a final concentration of 1% (w / v), incubated in a water bath for 1 h at 37 ° C. Next, the sample was centrifuged for 1 h at 200 ° C and 2000xg, the supernatant containing the hybrid protein was collected. The detergent was disposed of using the Detergent-OUTtmMicro Kit (Millipore), the detergent-free sample was concentrated on the SpeedVac unit to its original volume. Additional treatment of the final preparation was performed with 8 M urea in the presence of dithiotriethanol (0.02 M), followed by overnight dialysis against a carbonate buffer (pH 8.5).

The hybrid protein was sorbed (in carbonate buffer, pH 9.5–9.6) on a 96-well plate, kept overnight at 4 ° C. The tablet was treated with blocking buffer (0.01 M PBS, pH 7.2-7.4 with 5% ETS) for 1 h at room temperature, washed 3 times with PBS with Tween-20. 100 μl of 2-fold serum dilutions (starting at 1: 200) in blocking buffer were added to the wells of the plate, incubated for 1 h at room temperature. Serum was examined in duplicate. Polyclonal rabbit antibodies to Immunoglobulin A (NeoMarkers Inc., USA) at a dilution of 1: 7000 labeled with horseradish peroxidase were used as conjugates. TMB was used as a substrate. Accounting for the reaction was carried out at a wavelength of 450 nm. For the titer took the highest dilution of serum, which gives an optical density of at least 2 times more than that of non-immunized mice in the same dilution.

The results obtained indicate a high immunogenicity of the proteins obtained. When immunization with hybrid proteins in the blood of mice, antibodies were detected to all studied types of rotaviruses ranging from 1: 5200 in the case of rotavirus B, SEQ ID NO: 3, to 1: 70,000 in the case of rotavirus A,

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SEQ ID NO: 3 (table). The difference in antibody titers induced by the proteins of the group SEQ ID NO: 1, SEQ ID NO: 2, and the group SEQ ID NO: 3 - SEQ ID NO: 6 was not significant.

Thus, the formation of a multivalent immune response to rotaviruses of groups A, B and C in mice immunized with variants of the hybrid protein has been shown.

Example 8. The protectiveness of the immune response caused by hybrid proteins (SEQ ID NO: 6, SEQ ID NO: 5, SEQ ID NO: 2), against rotaviruses of groups A, B and C.

In the present study, Balb / c mice (females), 7-8 weeks (weighing 1719 g), obtained from the Institution of the Russian Academy of Sciences of the Institute of Bioorganic Chemistry named after Academicians M.M. Shemyakin and Yu.A. Ovchinnikova RAS (Branch) Nursery of laboratory animals Pushchino. Laboratory animals were clinically healthy and free from worms (veterinary certificate 302 No. 0207364 dated January 11, 2013). The animals were kept in the vivarium of the Federal State Research Institute of Pediatric Infections of the Federal Medical and Biological Agency of Russia in accordance with the applicable sanitary regulations.

Immunization of mice was performed intramuscularly and nasally: a hybrid protein was injected intramuscularly in the composition of the Freund's adjuvant (20 μg of the hybrid protein), an aqueous solution of the hybrid protein was nasally administered. Immunization was performed twice with an interval of 2 weeks.

To study the protection of the hybrid protein on the model of a lethal rotavirus infection, we used samples of rotaviruses A, B, and C provided by the Federal State Institution Scientific Research Institute of Pediatric Infections, FMBA Russia, adapted to mice. Titration of viruses to determine a single dose causing 50% of animal death was performed on Balb / c mice (females, 7-8 weeks old). The frozen virus was thawed, the murine lethal dose (causing 50% mortality) was determined by infecting mice with a tenfold dilution of the virus (3 mice per dilution). Observation of the death of the mice was carried out for 14 days after infection. The titer of the virus was determined by the method of Reed and Mench. For infection of mice, the virus was administered orally at a dose of 1 LD / 50 (the dose that causes the death of 50% of the mice) at 50 μl / mouse. After infection, animals were monitored daily for 14 days.

Infected mice with a sample of rotavirus A in the amount of 1.5-10 ⁸ plaque-forming units (PFU). The death of infected mice in the control group began on day 2, the survival rate of mice was 40%. In the experimental group (SEQ ID NO: 6), 64% survival of mice was observed (Fig. 1). The study of the dynamics of the body mass of the mice made it possible to establish that in the experimental group the decrease in body mass occurred more slowly and did not exceed 16%. In the control group, the body weight of mice decreased by 28% (Fig. 2).

The mice were infected with a sample of rotavirus B in an amount of 1.1-10 ⁷ plaque-forming units (PFU). The death of infected mice in the control group began on day 3, the survival rate of mice was 35%. In the experimental group (SEQ ID NO: 5), 45% survival of mice was observed (Fig. 3). The study of the dynamics of the body mass of the mice made it possible to establish that in the experimental group the decrease in body weight occurred more slowly and did not exceed 11%, since 10 days the body weight has increased. In the control group, the body weight of mice decreased by 22%, an increase in body weight in the studied period of time was not observed (Fig. 4).

Infected mice with a sample of rotavirus C in the amount of 1.2-10 ⁸ plaque-forming units (PFU). The death of infected mice in the control group also began on day 2, the survival rate of mice was 32%. In the experimental group (SEQ ID NO: 2), 68% survival of mice was observed (Fig. 5). The study of the dynamics of the body mass of the mice made it possible to establish that in the experimental group the decrease in body weight occurred more slowly and did not exceed 15%, from the 12th day we observed weight gain. In the control group, the body weight of mice decreased by 22% (Fig. 6).

Example 9. The protective effect of the vaccine based on chimeric protein (SEQ ID NO: 4), in a therapeutic model of a lethal infection with rotavirus groups A, B and C.

In the present study, Balb / c mice (pregnant females), 7-8 weeks old (weighing 18-21 g), obtained from the Institution of the Russian Academy of Sciences of the Institute of Bioorganic Chemistry. Academicians M.M. Shemyakin and Yu.A. Ovchinnikova RAS (Branch) Nursery of laboratory animals Pushchino. Laboratory animals were clinically healthy and free from worms (veterinary certificate 302 No. 0207364 dated January 11, 2013). The animals were kept in the vivarium of the Federal State Research Institute of Pediatric Infections of the Federal Medical and Biological Agency of Russia in accordance with the applicable sanitary regulations.

Infected newborn mice (10-day) with a sample of rotavirus A in the amount of 0.5-10 ⁴ plaque-forming units (PFU). On the same day, mice were injected with chimeric protein in an amount of 5 μg per mouse, with a physiologically acceptable carrier. Observed the survival of these mice within 14 days after the introduction of the lethal dose of rotavirus A.

When co-administered with rotavirus A and a mouse chimeric protein vaccine, they showed a survival rate of 80% a week after infection and 72% on day 14 of observation. In the control group, the survival rate was 0% already on day 5 of the experiment.

Infected mice with a sample of rotavirus B in an amount of 1.1-10 ³ plaque-forming units (PFU). On the same day, mice were injected with chimeric protein in an amount of 5 μg per mouse. Observed the survival of these mice within 14 days after the introduction of the lethal dose of rotavirus B.

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When co-administered with rotavirus B and a mouse chimeric protein vaccine, a survival rate of 76% was observed one week after infection and 66% on day 14 of observation. In the control group, the survival rate was 0% on the 8th day of the experiment.

Infected mice with a sample of rotavirus C in the amount of 1.2-10 ⁵ plaque-forming units (PFU). On the same day, mice were injected with chimeric protein in an amount of 5 μg per mouse. Observed the survival of these mice within 14 days after the introduction of the lethal dose of rotavirus C.

When co-administered with rotavirus C and a vaccine based on a mouse chimeric protein, a survival rate of 77% was observed one week after infection and 62% on the 14th day of observation. In the control group, the survival rate was 0% on the 10th day of the experiment.

The results obtained confirm the high immunogenicity of the two vaccines proposed in this invention, based on proteins of SEQ ID NO: 1 - SEQ ID NO: 6 and the possibility of their use for the treatment of rotavirus infection.

Vaccine for the prevention and treatment of rotavirus infection, containing a hybrid protein as an active agent (options)

Sequence Listing <110> RD-Biotech Limited Liability Company (RDBiotech LLC) <120> Vaccine for the prevention and treatment of rotavirus infection, containing a hybrid protein as an active agent (options) <150> RU 2013122473 <151> 2013 -05-15 <160> 6 <210> 1 <211> 175 <212> protein <213> Artificial sequence <400> 1

Met one Asp Val Leu Phe five Ser Leu Ser Lys Thr ten Leu Lys Asp Ala Arg 15 Asp Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln 20 25 thirty Gln Phe Asn Gln Met Ile Ile Thr Met Asn Gly Asn Asp Phe Gln Thr 35 40 45 Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe Gly 50 55 60 Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr 65 70 75 80 Ala Arg Thr Thr Ile Asp Tyr Phe Ile Asp Phe Val Asp Asn Val Cys 85 90 95 Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala Gly Gly 100 105 110 Gly Ser Gly Gly Gly Ser Thr Ile Asn Asn Asp Asn Ser Asn Val Ser 115 120 125 Ser Asp Ala Glu Phe Tyr Leu Ile Pro Gln Ser Gln Thr Ala Met Cys 130 135 140 Thr Gln Tyr Ile Asn Asn Gly Leu Pro Pro Ile Gln Asn Thr Arg Asn 145 150 155 160 Ile Val Pro Val Asn Ile Thr Ser Arg Gln Ile Lys Asp Ile Arg 165 170 175

<210> 2 <211> 175 <212> protein <213> Artificial sequence <400> 2

Met one Thr Ile Asn Asn five Asp Asn Ser Asn Val ten Ser Ser Asp Ala Glu 15 Phe Tyr Leu Ile Pro Gln Ser Gln Thr Ala Met Cys Thr Gln Tyr Ile Asn 20 25 thirty

- 15 031404

Asn glyu leu Pro Pro Ile Gln asn 40 Thr Arg Asn Ile Val 45 Pro Val Asn 35 Ile Thr Ser Arg Gln Ile Lys Asp Ile Arg Gly Gly Gly Ser Gly Gly 50 55 60 Gly Ser Asp Val Leu Phe Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg 65 70 75 80 Asp Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile 85 90 95 Gln Gln Phe Asn Gln Met Ile Ile Thr Met Asn Gly Asn Asp Phe Gln 100 105 110 Thr Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe 115 120 125 Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu 130 135 140 Thr Ala Arg Thr Thr Ile Asp Tyr Phe Ile Asp Phe Val Asp Asn Val 145 150 155 160 Cys Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala

165 170 175 <210> 3 <211> 459 <212> protein <213> Artificial sequence <400> 3

Met one Ala Gln val Ile five Asn Thr Asn Ser Leu ten Ser Leu Leu Thr Gln 15 Asn Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu 20 25 thirty Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35 40 45 Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala 50 55 60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly 65 70 75 80 Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala 85 90 95 Val Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile 100 105 110 Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly 115 120 125 Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu 130 135 140 Thr Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu 145 150 155 160 Lys Gln Ile Asn Ser Gln Thr Leu Gly Leu Gly Gly Gly Ser Gly Gly 165 170 175 Gly Ser Gly Gly Gly Ser Ala Ala Thr Thr Thr Glu Asn Pro Leu Gln 180 185 190 Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Thr Leu Arg Ser Asp Leu 195 200 205 Gly Ala Val Gln Asn Arg Phe Asn Ser Ala Ile Thr Asn Leu Gly Asn 210 215 220 Thr Val Asn Asn Leu Thr Ser Ala Arg Ser Arg Ile Glu Asp Ser Asp 225 230 235 240 Tyr Ala Thr Glu Val Ser Asn Met Ser Arg Ala Gln Ile Leu Gln Gln 245 250 255 Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro Gln Asn Val 260 265 270 Leu Ser Leu Leu Arg Gly Gly Gly Ser Gly Gly Gly Ser Asp Val Leu 275 280 285 Phe Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys Ile Val Glu 290 295 300 Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln Gln Phe Asn Gln

- 16 031404

305 310 315 320 Met Ile Ile Thr Met Asn Gly Asn Asp Phe Gln Thr Gly Gly Ile Gly 325 330 335 Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe Gly Leu Leu Gly Thr 340 345 350 Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr Ala Arg Thr Thr 355 360 365 Ile Asp Tyr Phe Ile Asp Phe Val Asp Asn Val Cys Met Asp Glu Met 370 375 380 Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Ser Thr Ile Asn Asn Asp Asn Ser Asn Val Ser Ser Asp Ala Glu 405 410 415 Phe Tyr Leu Ile Pro Gln Ser Gln Thr Ala Met Cys Thr Gln Tyr Ile 420 425 430 Asn Asn Gly Leu Pro Pro Ile Gln Asn Thr Arg Asn Ile Val Pro Val 435 440 445 Asn Ile Thr Ser Arg Gln Ile Lys Asp Ile Arg

450 455 <210> 4 <211> 459 <212> protein <213> Artificial sequence <400> 4

Met one Asp Val Leu Phe five Ser Leu Ser Lys Thr ten Leu Lys Asp Ala Arg 15 Asp Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln 20 25 thirty Gln Phe Asn Gln Met Ile Ile Thr Met Asn Gly Asn Asp Phe Gln Thr 35 40 45 Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe Gly 50 55 60 Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr 65 70 75 80 Ala Arg Thr Thr Ile Asp Tyr Phe Ile Asp Phe Val Asp Asn Val Cys 85 90 95 Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala Gly Gly 100 105 110 Gly Ser Gly Gly Gly Ser Thr Ile Asn Asn Asp Asn Ser Asn Val Ser 115 120 125 Ser Asp Ala Glu Phe Tyr Leu Ile Pro Gln Ser Gln Thr Ala Met Cys 130 135 140 Thr Gln Tyr Ile Asn Asn Gly Leu Pro Pro Ile Gln Asn Thr Arg Asn 145 150 155 160 Ile Val Pro Val Asn Ile Thr Ser Arg Gln Ile Lys Asp Ile Arg Gly 165 170 175 Gly Gly Ser Gly Gly Gly Ser Ala Gln Val Ile Asn Thr Asn Ser Leu 180 185 190 Ser Leu Leu Thr Gln Asn Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly 195 200 205 Thr Ala Ile Glu Arg Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys 210 215 220 Asp Asp Ala Ala Gly Gln Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile 225 230 235 240 Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile 245 250 255 Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln 260 265 270 Arg Val Arg Glu Leu Ala Val Gln Ser Ala Asn Ser Thr Asn Ser Gln 275 280 285 Ser Asp Leu Asp Ser Ile Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu

- 17 031404

290 295 300 Ile Asp Arg Val Ser Gly Gln Thr Gln Phe Asn Gly Val Lys Val Leu 305 310 315 320 Ala Gln Asp Asn Thr Leu Thr Ile Gln Val Gly Ala Asn Asp Gly Glu 325 330 335 Thr Ile Asp Ile Asp Leu Lys Gln Ile Asn Ser Gln Thr Leu Gly Leu 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Thr Thr 355 360 365 Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val Asp 370 375 380 Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser Ala 385 390 395 400 Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser Ala Arg Ser 405 410 415 Arg Ile Glu Asp Ser Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Arg 420 425 430 Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn 435 440 445 Gln Val Pro Gln Asn Val Leu Ser Leu Leu Arg

450 455 <210> 5 <211> 459 <212> protein <213> Artificial sequence <400> 5

Met one Thr Ile Asn asn five Asp Asn Ser Asn Val ten Ser Ser asp Ala glu 15 Phe Tyr Leu Ile Pro Gln Ser Gln Thr Ala Met Cys Thr Gln Tyr Ile Asn 20 25 thirty Asn Gly Leu Pro Pro Ile Gln Asn Thr Arg Asn Ile Val Pro Val Asn 35 40 45 Ile Thr Ser Arg Gln Ile Lys Asp Ile Arg Gly Gly Gly Ser Gly Gly 50 55 60 Gly Ser Asp Val Leu Phe Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg 65 70 75 80 Asp Lys Ile Val Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile 85 90 95 Gln Gln Phe Asn Gln Met Ile Ile Thr Met Asn Gly Asn Asp Phe Gln 100 105 110 Thr Gly Gly Ile Gly Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe 115 120 125 Gly Leu Leu Gly Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu 130 135 140 Thr Ala Arg Thr Thr Ile Asp Tyr Phe Ile Asp Phe Val Asp Asn Val 145 150 155 160 Cys Met Asp Glu Met Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala Gly 165 170 175 Gly Gly Ser Gly Gly Gly Ser Ala Gln Val Ile Asn Thr Asn Ser Leu 180 185 190 Ser Leu Leu Thr Gln Asn Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly 195 200 205 Thr Ala Ile Glu Arg Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys 210 215 220 Asp Asp Ala Ala Gly Gln Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile 225 230 235 240 Lys Gly Leu Thr Gln Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile 245 250 255 Ala Gln Thr Thr Glu Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln 260 265 270 Arg Val Arg Glu Leu Ala Val Gln Ser Ala Asn Ser Thr Asn Ser Gln

- 18 031404

275 280 285 Ser Asp Leu Asp Ser Ile Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu 290 295 300 Ile Asp Arg Val Ser Gly Gln Thr Gln Phe Asn Gly Val Lys Val Leu 305 310 315 320 Ala Gln Asp Asn Thr Leu Thr Ile Gln Val Gly Ala Asn Asp Gly Glu 325 330 335 Thr Ile Asp Ile Asp Leu Lys Gln Ile Asn Ser Gln Thr Leu Gly Leu 340 345 350 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Ala Ala Thr Thr 355 360 365 Thr Glu Asn Pro Leu Gln Lys Ile Asp Ala Ala Leu Ala Gln Val Asp 370 375 380 Thr Leu Arg Ser Asp Leu Gly Ala Val Gln Asn Arg Phe Asn Ser Ala 385 390 395 400 Ile Thr Asn Leu Gly Asn Thr Val Asn Asn Leu Thr Ser Ala Arg Ser 405 410 415 Arg Ile Glu Asp Ser Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Arg 420 425 430 Ala Gln Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn 435 440 445 Gln Val Pro Gln Asn Val Leu Ser Leu Leu Arg

450 455 <210> 6 <211> 459 <212> protein <213> Artificial sequence <400> 6

Met one Ala Gln val Ile five Asn Thr Asn Ser Leu ten Ser Leu Leu Thr Gln 15 Asn Asn Leu Asn Lys Ser Gln Ser Ala Leu Gly Thr Ala Ile Glu Arg Leu 20 25 thirty Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln 35 40 45 Ala Ile Ala Asn Arg Phe Thr Ala Asn Ile Lys Gly Leu Thr Gln Ala 50 55 60 Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly 65 70 75 80 Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ala 85 90 95 Val Gln Ser Ala Asn Ser Thr Asn Ser Gln Ser Asp Leu Asp Ser Ile 100 105 110 Gln Ala Glu Ile Thr Gln Arg Leu Asn Glu Ile Asp Arg Val Ser Gly 115 120 125 Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ala Gln Asp Asn Thr Leu 130 135 140 Thr Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Asp Ile Asp Leu 145 150 155 160 Lys Gln Ile Asn Ser Gln Thr Leu Gly Leu Gly Gly Gly Ser Gly Gly 165 170 175 Gly Ser Gly Gly Gly Ser Ala Ala Thr Thr Thr Glu Asn Pro Leu Gln 180 185 190 Lys Ile Asp Ala Ala Leu Ala Gln Val Asp Thr Leu Arg Ser Asp Leu 195 200 205 Gly Ala Val Gln Asn Arg Phe Asn Ser Ala Ile Thr Asn Leu Gly Asn 210 215 220 Thr Val Asn Asn Leu Thr Ser Ala Arg Ser Arg Ile Glu Asp Ser Asp 225 230 235 240 Tyr Ala Thr Glu Val Ser Asn Met Ser Arg Ala Gln Ile Leu Gln Gln 245 250 255 Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro Gln Asn Val 260 265 270

- 19 031404

Leu Ser Leu 275 Leu Arg Gly Gly Gly 280 Ser Gly Gly Gly Ser 285 Thr Ile Asn Asn Asp Asn Ser Asn Val Ser Ser Asp Ala Glu Phe Tyr Leu Ile Pro 290 295 300 Gln Ser Gln Thr Ala Met Cys Thr Gln Tyr Ile Asn Asn Gly Leu Pro 305 310 315 320 Pro Ile Gln Asn Thr Arg Asn Ile Val Pro Val Asn Ile Thr Ser Arg 325 330 335 Gln Ile Lys Asp Ile Arg Gly Gly Gly Ser Gly Gly Gly Ser Asp Val 340 345 350 Leu Phe Ser Leu Ser Lys Thr Leu Lys Asp Ala Arg Asp Lys Ile Val 355 360 365 Glu Gly Thr Leu Tyr Ser Asn Val Ser Asp Leu Ile Gln Gln Phe Asn 370 375 380 Gln Met Ile Ile Thr Met Asn Gly Asn Asp Phe Gln Thr Gly Gly Ile 385 390 395 400 Gly Asn Leu Pro Ile Arg Asn Trp Asn Phe Asp Phe Gly Leu Leu Gly 405 410 415 Thr Thr Leu Leu Asn Leu Asp Ala Asn Tyr Val Glu Thr Ala Arg Thr 420 425 430 Thr Ile Asp Tyr Phe Ile Asp Phe Val Asp Asn Val Cys Met Asp Glu 435 440 445 Met Ala Arg Glu Ser Gln Arg Asn Gly Ile Ala 450 455

Claims (7)

CLAIM 1. A vaccine for the prevention and treatment of rotavirus infection, containing an effective amount as an active agent of a hybrid protein, including rotavirus polypeptides, and a physiologically acceptable carrier, characterized in that the hybrid protein is characterized by the sequence SEQ ID NO: 1 or SEQ ID NO: 2, and rotavirus polypeptides are represented by an immunogenic epitope of the VP8 protein and immunogenic epitopes of the VP6 protein and are connected by a flexible bridge. 2. The vaccine according to claim 1, wherein the hybrid protein additionally contains a fragment of flagellin, which is represented by the epitopes FliC1, FliC2, connected to the polypeptides by a flexible bridge, and the hybrid protein is characterized by the sequence SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. 3. The vaccine according to claim 1 or 2, characterized in that the active agent is obtained by a method that includes creating a codon-optimized composition for expression in the producer cells of a recombinant DNA encoding a hybrid protein, introducing DNA into a vector construct that ensures a high level of its expression in cells the producer, the introduction of this vector construct into the producer cells, the isolation of the synthesized fusion protein, its purification and mixing with a physiologically acceptable carrier. 4. Vaccine according to claim 3, characterized in that prokaryotic cells are used as producer cells. 5. Vaccine according to claim 3, characterized in that eukaryotic cells are used as producer cells. 6. The vaccine according to claim 3, characterized in that the method for producing the active agent further comprises introducing a vector construct into a carrier cell, delivering it to the producer cells. 7. Vaccine according to claim 6, characterized in that cells of higher plants are used as producer cells. - 20 031404 FIG. one FIG. 2 FIG. 3 - 21 031404 4 ^ j)