RU2789354C1 - Method for obtaining virus-like particles of norovirus containing the protein vp1 of enterovirus echovirus 30, in vitro - Google Patents

Abstract

FIELD: molecular virology.

SUBSTANCE: invention relates to the field of molecular virology, biotechnology and medicine, in particular to the production of a chimeric protein that spontaneously forms in vitro virus-like particles (VLP) containing the protein VP1 Echovirus 30 (ECHO30). The obtained virus-like norovirus particles containing the protein VP1 of the enterovirus Echovirus 30 on their surface can be used to prepare vaccines against non-polio enteroviruses. The method for obtaining virus-like particles of norovirus containing the protein VP1 Echovirus 30 (ECHO30) in vitro includes cultivation of E. coli cells transformed by chimeric protein in a suitable culture medium, isolation and purification of virus-like particles. In this case, a chimeric protein S_N-VP1_E30 is created with a size of 516 amino acids with a molecular weight of 57 kDa, with a structure in accordance with the sequence SEQ ID NO: 1 and the transformation of E. coli cells, Rosette strain 2 (DE3) with a plasmid containing a nucleotide sequence encoding the amino acid sequence SEQ ID NO: 1.

EFFECT: implementation of the invention provides for the creation of a chimeric protein SN-VP1E30 with a size of 516 amino acids with a molecular weight of 57 kDa, which is capable of spontaneously assembling into VLP presenting antigenic determinants of the protein VP1 of enterovirus E30.

1 cl, 4 dwg, 6 ex

Description

The invention relates to the field of molecular virology, biotechnology and medicine, in particular to the production of a chimeric protein that spontaneously forms in vitro virus-like particles (VLP) containing the VP1 protein of the enterovirus Echovirus 30 (Picornaviridae: Enterovirus: Enterovirus B), and can be used to prepare vaccines for the prevention of enteroviral meningitis.

Enteroviruses (EV) (genus Enterovirus, family Picornaviridae, order Picornavirales) are a large group of non-enveloped RNA-containing viruses that can cause diseases of various clinical presentation and severity, mainly in children of the first years of life. Enterovirus infection can manifest itself in the form of exanthema of the mouth and extremities (hand, foot and mouth disease - HFMD), herpangina, meningitis, meningo-encephalitis, encephalitis, polyneuropathy, flaccid paralysis, myocarditis, etc. The severity of the course of the disease can vary from mild fever to multisystem manifestations with damage to the cardiovascular and central nervous systems. Often, enterovirus infections have an epidemic distribution, covering large groups of the child population [Fomina S.G., Novikova N.A. Enteroviruses in children with gastroenteritis // Medial. - 2014. - No. 12 (2). - S. 58-71.; Mao Q. EV-A71 vaccine license: a first step for multivalent enterovirus vaccine to control HFMD and other severe diseases/ Q. Mao, Y. Wang, L. Bian, M. Xu, Z. Liang//Emerging Microbes and Infections - 2016, V.5.7.].

On the basis of genetic characteristics and antigenic properties, the enteroviruses are divided into four types (A - D), among which viruses belonging to the Enterovirus A (EV-A) and Enterovirus B (EV-B) species are most common [Zell R. ICTV virus taxonomy profile: picornaviridae/ R. Zell, E. Delwart, A.E. Gorbalenya, T. Hovi, A.M.Q. King, N.J. Knowles//J. Gen. Virol. - 2017. Vol. 98. 10. - P. 2421-2.]. Enteroviruses of the EV-A type (enterovirus A71, Coxsackie A6, Coxsackie A10, Coxsackie A16, etc.) most often cause HFMD in young children. The vast majority of patients recover completely in 7-10 days. However, some patients develop serious complications caused by damage to the central nervous system, single lethal cases are described. Representatives of the EV-B species (Coxsackie B3, Coxsackie B5, ECHO25, ECHO30 viruses) are more often associated with serious diseases such as myocarditis, aseptic meningitis, encephalitis and hepatitis, often leading to disability or death [Xu W. Distribution of enteroviruses in hospitalized children with hand, foot and mouth disease and relationship between pathogens and nervous system complications/W. Xu, C. Liu, L. Yan, J. Li, L. Wang, Y. Qi//Virology J. - 2012. 9. (8).]. Among EV-B, ECHO viruses stand out because they cause aseptic meningitis and encephalitis not only in children, but also in adults [Kupila L. Etiology of aseptic meningitis and encephalitis in an adult population/L. Kupila, T. Vuorinen, R. Vainionpaa, V. Hukkanen, R.J. Marttila., P.//Neurology. - 2006. Vol. 66. - P. 75-80.].

ECHO viruses are characterized by the epidemic nature of the incidence, expressed in some cases in the registration of hundreds, thousands and millions of patients. The most frequent are outbreaks of serous meningitis, the causative agents of which in Europe, Asia, and America are predominantly ECHO 11 and 30 viruses. . The process of intrapopulation variability and selection of epidemic variants, which cannot be observed and controlled, is realized in some cases by the "sudden" development of outbreaks characterized by lesions unusual for these viruses in any geographical areas. The virus multiplies and accumulates in the lymphoid tissue of the nasopharynx and gastrointestinal tract, causing local pathological changes in the mucous membranes of the respiratory tract and intestines. The virus is released from the nasopharynx within a week, and from the intestines - up to a month. In the case of a transition to the stage of viremia, the infection penetrates the central nervous system, affecting the substance of the brain and spinal cord, pia mater, as well as the myocardium and pericardium, pancreas, muscle tissue, liver, eye tissues, and skin. Type-specific virus-neutralizing antibodies appear in the blood serum of patients along with the first symptoms of the disease.

Enteroviruses are ubiquitous, however, in the countries of Southeast Asia, EV-A has the greatest epidemic significance, in countries located in Europe and North America, EV-B [Chang L.Y Enterovirus A71 neurologic complications and long-term sequelae/ L.Y. Chang, H.Y. Lin, S.S. Gau, C.Y. Lu, S.H. Hsia, Y.C. Huang, et al.//J. Biomed. sci. - 2019. 26(57); Abedi G.R. Enterovirus and human parechovirus surveillance - United States, 2009-2013/G.R. Abedi, J.T. Watson, H. Pham, W.A. Nix, M.S. Oberste, S.I. Gerber // Morbidity and Mortality Weekly Report. - 2015. Vol. 64. No. 34. - P. 940-943.]. Epidemiological studies of the prevalence of enteroviruses in the territory of the Russian Federation have shown that recently in children and adults with enteroviral meningitis, the ECHOZO virus is most often detected [Golitsyna L.N., Zverev V.V., Selivanova S.G., Ponomareva N. V., Kashnikov A.Yu., Sozonov D.V., et al. Etiological structure of enterovirus infections in the Russian Federation in 2017-2018//ZNIISO. - 2019. - No. 8. - S. 30-8.].

More than 60 of the 102 currently known enterovirus serotypes cause several dozen forms of disease, the most severe of which are enteroviral meningitis (EVM), meningoencephalitis, viral sepsis and neonatal liver necrosis, uveitis, myocarditis, pancreatitis, hepatitis, lethal pulmonary edema [Rhoades, R.E. Enterovirus Infections of the Central Nervous System/R.E. Rhoades, J.M. Tabor-Godwin, G. Tsueng and R. Feuer//Virology. - 2011. Vol.411. No. 2. - P. 288-305.; A.H. Lukashev, L.H. Golitsyna, Yu.A. Vakulenko, L.V. Akhmadishina, N.I. Romanenkova, E.Yu. Sapega, N.S. Morozova, N.A. Novikova, O.E. Trotsenko, O.E. Ivanova Modern possibilities and directions of development of molecular epidemiological monitoring in the surveillance of enteroviral infections. Experience of the Russian Federation//Infection and Immunity - 2018. - V. 8. No. 4. - S. 452-464.].

Vaccination is only available for polioviruses. The variety of serotypes of non-polio enteroviruses and their genetic variability make it difficult to develop vaccines against this group of pathogens. Enterovirus 71 vaccines are currently in clinical trials, but none of the proposed candidate vaccines has yet passed all stages of testing.

A promising direction in the field of vaccination against (incompletely) enteroviruses is the development of vaccines based on virus-like particles.

Virus-like particles (VLPs) resemble mature virions but do not contain the genetic material of the virus. VLPs mimic the structure of the viral capsid but do not contain genes, so they cannot replicate or provide a mechanism for secondary infection. VLPs are assembled after expression of specific viral proteins and are located on the outer surface, resembling the outer surface of the corresponding virus, but unlike a true viral particle, they do not include genetic material and are optimal delivery systems for the presentation of neutralizing epitopes to the immune system. With the ability to interact effectively with antigen-presenting cells, VLPs maximize the immunogenic potential of neutralizing epitopes and the protective properties of antibodies [Mohsen M.O. Interaction of viral capsid-derived virus-like particles (VLPs) with the innate immune system/ M.O. Mohsen, A.C. Gomes, M. Vogel, M.F. Bachmann//Vaccines. - 2018. - Vol. 6. No. 3. - P. 37.].

In VLP vaccines, the conformational structure of epitopes is preserved, which is important for the immunogenicity of vaccines, in addition, there is no risk of obtaining virulent revertants, which occurs when using live attenuated vaccines.

To date, a number of VLP vaccines (for example, against HIV, coronaviruses) have been designed that cause the formation of virus-neutralizing antibodies and stimulate T-cell cytotoxic response. Yes, Y.C. Hu et al. [Hu Y.C. Formation of enterovirus-like particle aggregates by recombinant baculoviruses co-expressing P1 and 3CD in insect cells/Y.C. Hu, J.T. Hsu, J.H. Huang, M.S. Ho, Y.C. Ho//Biotechnol Lett. - 2003. Vol. 25. No. 12. - P. 919-925.] and Y.C. Chung et al. [Chung Y.C.. Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protects mice against lethal chal-lenge/Y.C. Chung, M.S. Ho, W.-J. Chen, J.-H. Huang, S.T. Chou Y., Ch. Hu//Vaccines. - 2008. Vol. 26. No. 15. - P. 1822-1862.] used a recombinant insect baculovirus system to produce VLP-EV 71, which elicited Th-1 and Th-2 helper responses in animal experiments. At the same time, it was shown that the offspring obtained from those immunized with the vaccine were protected from infection with enterovirus 71.

Currently, VLP vaccines are mainly developed using insect cells, which limits the scale of vaccine production. Transgenic plants and yeasts capable of producing VLP particles may be useful for this technology, making them suitable for oral vaccination or injection. However, when EV VLPs are produced in vivo in insect or yeast cells for use as an antigen in vaccines, the capsid proteins assemble into VLPs within host cells, resulting in encapsulation of host-cell-associated contaminants within the VLP, potentially capable of eliciting unwanted immune responses. response, including allergic reactions [Le D.T. In Vitro Assembly of virus-like particles and their applications/D.T.Le, K.M. Müller//Life. - 2021. Vol. 11. No. 4.: 334.].

In subsequent studies, recombinant technologies were developed to obtain and study virus-like particles of enterovirus 71 [Chung Y.C., But M.S., Wu J.C., Chen W.J., Huang J.H., Chou S.T. et al. Immunization with virus-like particles of enterovirus 71 elicits potent immune responses and protect mice against lethal challenge. Vaccine. 2008; 26: 1855-1862.] and combinations of EV 71 and Coxsackievirus A 16 (bivalent vaccines) [Zhao H. Novel recombinant chimeric virus-like particle is immunogenic and protective against bother enterovirus 71 coxsackievirus A16 in mice/H.Zhao, H. -Ya Li, Ji.-F. Han, Yo.-Q., Deng Sh.-Ya. Zhu, X.-F. Li. et al.// Scientific Reports. - 2015. Vol. 5. No. 1. - P. 1-8.]. These drugs activated Th1 and Th2 cell immune responses and were effective when tested in the mouse passive defense test.

As the closest analogue, we consider a substituted norovirus capsid protein monomer having only a P-domain, containing a foreign antigen introduced into one or more of the three surface loops present on each P-domain monomer by molecular cloning. The recombinant monomer is easily formed in Escherichia coli and yeast, is highly stable and resistant to a wide range of physicochemical conditions [US patent No. 9096644, 04.08.2015].

Noroviruses are a genus of RNA viruses belonging to the Caliciviridae family. There are five genogroups of noroviruses (GI-GV). The most common is the second genogroup (GII), which accounts for up to 80-90% of all cases of norovirus gastroenteritis. Viruses are highly contagious. Infection with noroviruses causes the appearance of specific antibodies that form an immune response against the pathogen and prevent re-infection, but immunity is temporary. There is a genetically determined immunity to norovirus infection (up to 15% in the population) and the possibility of an asymptomatic course (up to 10-13% in the population). The causative agent has a pronounced variability, this is associated with a high probability of reinfection due to the impossibility of forming a stable immunity to it. Viral protein 1 (VP1) of norovirus is the main structural component of the capsid surface and consists of two domains - S (shell domain), responsible for the formation of the capsid, and P (protruding), carrying the receptor binding site. A platform based on the norovirus VP1 protein has previously been proposed for the presentation of viral antigens to the immune system. It was shown that proteins derived from E. coli, consisting of the amino acid sequence of the S domain of VP1 fused into one molecule with fragments of the VP8 amino acid sequences of the rotavirus, influenza A (H7N9) virus hemagglutinin, or the hepatitis E virus capsid protein, are able to form VLPs containing on surface added peptides. Immunization of laboratory animals with chimeric VLPs led to the production of neutralizing antibodies in high titers [Xia M. Bioengineered norovirus S60 nanoparticles as a multifunctional vaccine platform/ M., P. Huang, C. Sun, L. Han, F.S. Vago, K. Li, et al.// ACS Nano. - 2018. Vol. 12: #11. - P. 1-20; Tan M. Norovirus P particle, a novel platform for vaccine development and antibody production/M., P. Huang, M. Xia, P.A. Fang, W. Zhong, M. McNeal, et al. / / J Virol. - 2011. Vol. 85. No. 2. - P. 753-64.].

The objectives to be solved by the invention are the development of the structure of a recombinant chimeric protein consisting of amino acid sequences of the VP1 protein of norovirus fam. Caliciviridae and the VP1 protein of the enterovirus Echovirus 30 fam. Picornaviridae fused into one molecule and development of a method for obtaining virus-like norovirus particles containing on their surface the VP1 protein of the enterovirus Echovirus 30 (E30) in E. coli cells, strain Rosetta2 DE3.

The technical result of the invention is to create a chimeric protein S _N -VP1 _E30 with a size of 516 amino acids, with a molecular weight of 57 kDa, which is capable of spontaneously assembling into VLPs presenting antigenic determinants of the VP1 protein of enterovirus E30. In the primary structure of norovirus RNA, the site encoding the S and hinge regions of the VP1 protein (S _N ) is determined by bioinformatics methods.

The technical result achieved in the implementation of the claimed invention consists in the development of a method for obtaining virus-like norovirus particles containing on their surface the VP1 protein of enterovirus E30, including the manufacture of a genetic construct, which in the plasmid DNA contains the sequence SEQ ID NO: 1 encoded by the chimeric protein S _N -VP1 _E30 which is used to transform E. coli cells, strain Rosetta2 DE3.

There is no information in the prior art indicating the possibility of obtaining virus-like particles containing the VP1 E30 protein by means of the S _N -VP1 _E30 chimeric protein based on the nucleotide sequences of the genes encoding the norovirus VP1 protein and the E30 VP1 protein, consisting of sequences encoding Shell(S) and the hinge regions of the norovirus VP1 protein and the VP1 E30 protein, transformation of E. coli cells with a chimeric protein, followed by cultivation in a suitable culture medium and isolation of S _N -VP1 _E30 by affinity chromatography and in vitro self-assembly of VLP from S _N -VP1 _E30 . The method is carried out as follows way.

Create a chimeric protein S _N -VP1 _E30 size of 516 amino acids with a molecular weight of 57 kDa with the structure in accordance with the sequence of SEQ ID NO: 1:

To create a chimeric protein in the primary structure of norovirus RNA, bioinformatics methods determine the region encoding the S and hinge regions of the VP1 protein (S _N ). In the structure of the E30 virus RNA, the region encoding the VP1 protein (VP1 _E30 ) is determined. These sites are combined into one molecule to form a chimeric nucleotide sequence S _N -VP1 _E30 , add a sequence encoding 6 histidines and a stop codon. The resulting nucleotide sequence is used to predict the amino acid. In the chimeric sequence, sites encoding amino acids located at positions 57, 58, and 136 are replaced with nucleotides encoding cysteine. Next, the codons are optimized for expression in E. coli. The recombinant protein is expressed using the E. coli Rosetta 2 (DE3) strain (Novagen, USA). E. coli cells are transformed with a plasmid containing the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

The essence of the claimed invention is illustrated by drawings, where:

in FIG. 1 shows the structural organization of the S _N -VP1 _E30 chimeric protein,

in FIG. 2 shows the results of purification of S _N -VP1 _E30 from E. coli,

in FIG. 3 shows the electrophoretic mobility of undenatured (1) and denatured S _N -VP1 _E30 ,

in FIG. 4 shows VLPs generated by S _N -VP1 _E30 in vitro.

Implementation of the invention.

Example 1 Construction of a fusion protein.

Nucleotide sequences (n.p.) of the genes of the VP1 protein of the norovirus GII.4 genotype (GenBank no. MZ958411) and the VP1 protein of the ECHO30 genotype h (GenBank no. KP090772) of strains circulating in Russia were used in this work. The fusion gene was constructed based on nucleotide sequence analysis using the Lasergene software package (Dnastar, Inc.). Codon optimization was performed based on information provided in the Codon usage database (http://www.kazusa.or.jp/codon/). DNA was synthesized at Lumiprob RUS (Russia) and cloned into pET22b (Novagen, United States).

Example 2 Expression and purification of a recombinant protein.

The recombinant protein was expressed using the E. coli Rosetta 2 (DE3) strain (Novagen, United States). E. coli cells were transformed with a plasmid encoding the recombinant protein using an Eppendorf multiporator (Germany). E. coli were grown at 37°C in LB medium (1% tryptone, 2% yeast extract, 0.9% NaCl and 100 μg/ml ampicillin) to an optical density of 0.7. Expression was induced by adding isopropyl-β-D-1-thiogalactopyranoside to a concentration of 0.5 mm, after which the culture was incubated for 6 hours at 30°C. Cells were pelleted by low speed centrifugation, washed once in 0.9% NaCl, and lysed using the BugBuster Protein Extraction Reagent (Novagen, USA) according to the manufacturer's recommendations. The lysate was separated by centrifugation at 12000 g for 15 min into soluble and precipitated protein fractions. To purify the recombinant protein, the precipitated fraction was dissolved in a buffer containing 50 mM Tris-HCl, 6 M guanidine hydrochloride, 150 mM NaCl, pH 7.5, and then affinity chromatography was performed using IMAC Sepharose 6 Fast Flow (GE Healthcare, Sweden) according to the recommendations manufacturer for denaturing conditions. Protein renaturation was performed by dialysis against 3000 volumes of buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20%, 10%, or 5% sucrose.

Example 3 Determination of protein concentration.

The protein concentration in the solution was determined on a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) at a wavelength of 280 nm. The efficiency of protein renaturation was assessed by comparing the concentration of soluble protein before and after dialysis. The protein concentration before dialysis was taken as 100%.

Example 4 PAGE electrophoresis and immunoblotting.

All stages of expression and purification of the recombinant protein were controlled by protein electrophoresis in 10% PAAG in the presence of sodium dodecyl sulfate. The results were visualized by staining the gel with Coomassie brilliant blue R250. The molecular weight marker Thermo Scientific PageRuler Unstained Protein Ladder (10 to 200 kDa) was used. For immunoblotting, proteins were transferred from the gel onto a PVDF membrane (Thermo Scientific, USA) using a wet transfer system (Bio-Rad, USA). PageRuler Plus Prestained Protein Ladder (Thermo Scientific, USA) was used to control the transfer and determine the molecular weight. The membrane was blocked with 1% bovine serum albumin in phosphate buffered saline and incubated with anti-His-HRP:GG1-6F4.3.2 antibodies (Miltenyi Biotec, USA). The membrane was stained in a solution of Super Signal West Dura Extended Duration Substrate (Thermo Scientific, USA) and chemiluminescence was measured using a C-DiGit Blot Scanner (Li-Cor, USA) according to the manufacturer's recommendations.

Example 5: Self-assembly of VLP.

Self-assembly of the VLP from the chimeric protein was performed in vitro by dialysis against 3000 volumes of buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20%, 10% or 5% sucrose. The results of self-assembly were evaluated by EP in PAAG and electron microscopy. Transmission electron microscopy was used. An electron microscopic copper grid coated with a parlodium film was coated with 5 μl of a protein solution at a concentration of 100 μg/ml, washed with water to remove unbound components, and stained with an aqueous solution of 2% uranyl acetate (pH 4.5). The samples were examined using an HT7700 transmission electron microscope (Hitachi, Japan).

Example 6. Characterization of the antigenic properties of VP1 enterovirus Echovirus 30 in the PTR.

Antigenic properties were studied by enzyme immunoassay. To do this, PTP was diluted in physiological saline and adsorbed in a 96-well plate, incubated for 24 hours. 10 µl of human blood plasma known to contain antibodies to VP1 Echovirus 30 were added to the wells of a plate containing 90 µl of phosphate-buffered saline solution with tween (PBS-T). 500 rpm After incubation, the wells of the plate were washed 5 times with 1x PBS-T. After washing, the remaining moisture was removed and 100 μl of a conjugate solution (140 ng/ml) was added to all wells, which is monoclonal antibodies to IgG produced by clone 3D3cc, labeled with horseradish peroxidase (MoAbs to IgG HPR) manufactured by Hytest (Russia), and incubated 60 minutes in a shaker at 37°C. After incubation, washing with 1x PSB-T was repeated five times. Next, 100 μl of Hytest TMB solution (Russia) was added to all wells and incubated for 10 minutes, avoiding sunlight. The reaction was stopped with 50 μl of 1N sulfuric acid and the optical density was measured on an Infinite M200 Pro spectrophotometer (Tecan, Austria) in a two-wave mode: at a fundamental wavelength of 450 nm and a reference wavelength of 680 nm. The obtained data were analyzed using Magellan 7.2 (Tecan, Austria) and Microsoft Excel (Microsoft, USA) software.

The conducted studies have shown the feasibility of the claimed method for obtaining in vitro virus-like particles consisting of a chimeric recombinant protein, including the S-domain and the hinge part of the norovirus VP1 protein, fused with the full-length VP1 protein of the ECHO30 enterovirus.

After induction, E. coli produced an additional protein containing a sequence of 6 histidines, which confirmed the expression of SN-VP1 _E30 . When comparing the set of E. coli proteins in the soluble and sediment fractions, it was found that the sediment fraction contained a large amount of protein with an approximate molecular weight of 50–60 kDa. The calculated mass of S _N -VP1 _E30 was 57 kDa, and after dissolving the sediment fraction of proteins in a buffer containing 6 M guanidine hydrochloride, S _N -VP1 _E30 containing a histidine tail was isolated by affinity chromatography. The electrophoretic mobility of the purified S _N -VP1 _E30 coincided with the calculated one.

The ability of S _N -VP1 _E30 monomers to self-assemble into VLPs was examined by electron microscopy. The preparation used was S _N -VP1 _E30 renatured in the presence of 20% sucrose, in which spherical virus-like particles of approximately 50 nm in size were found. . Preservation of the antigenic properties of VP1 Echovirus 30 in the VLP was assessed by its interaction with antibodies to VP1 Echovirus 30 present in human plasma.

--->

Sequence listing

˂110˃ Federal budgetary institution of science "Nizhny Novgorod Scientific and

Research Institute of Epidemiology and Microbiology. academician I.N.

Blokhina of the Federal Service for Supervision of Consumer Rights Protection and

human well-being (FBUN NNIIEM named after academician I.N. Blokhina

Rospotrebnadzor)

˂120˃ Method for obtaining virus-like particles of norovirus ( Caliciviridae:

Norovirus ) containing the VP1 protein of the enterovirus Echovirus 30 ( Picornaviridae:

Enterovirus: Enterovirus B ), in vitro

˂160˃ 1

˂210˃ 1

˂211˃ 516

˂212˃PRT

˂213˃ Artificial Sequence

<220>

˂223˃ Chimeric protein consisting of the amino acid sequences of the VP1

protein of the norovirus (Caliciviridae: Norovirus) and the VP1 protein of

the enterovirus Echovirus 30 (Picornaviridae: Enterovirus: Enterovirus B)

fused into one molecule

<300>

<301> Novikov, Dmitry V.; Melentev, Dmitry A.; Mokhonov, Vladislav V.;

Kashnikov, Alexander Yu.; Novikova, Nadezhda A.; Lapin, Vladislav A.;

Mokhonova, Ekaterina V.; Novikov, Viktor V.

<302> Construction of norovirus ( Caliciviridae: Norovirus ) virus-like

particles containing VP1 of the Echovirus 30 ( Picornaviridae: Enterovirus:

Enterovirus B )

<303> Problems of Virology

<304> 66

<305> 5

<306> 383-389

<307> 2021-10-31

˂400˃ 1

Met Lys Met Ala Ser Ser Asp Ala Asn Pro Ser Asp Gly Ser Ala

1 5 10 15

Ala Asn Leu Val Pro Glu Val Asn Asn Glu Val Met Ala Leu Glu

20 25 30

Pro Val Val Gly Ala Ala Ile Ala Ala Pro Val Ala Gly Gln Gln

35 40 45

Asn Val Ile Asp Pro Trp Ile Arg Asn Asn Phe Cys Cys Ala Pro

50 55 60

Gly Gly Glu Phe Thr Val Ser Pro Ala Asn Ala Pro Gly Glu Ile

65 70 75

Leu Trp Ser Ala Pro Leu Gly Pro Asp Leu Asn Pro Tyr Leu Ser

80 85 90

His Leu Ala Arg Met Tyr Asn Gly Tyr Ala Gly Gly Phe Glu Val

95 100 105

Gln Val Ile Leu Ala Gly Asn Ala Phe Thr Ala Gly Lys Ile Ile

110 115 120

Phe Ala Ala Val Pro Pro Asn Phe Pro Thr Glu Gly Leu Ser Pro

125 130 135

Cys Gln Val Thr Cys Phe Pro His Ile Ile Val Asp Val Arg Gln

140 145 150

Leu Glu Pro Val Leu Ile Pro Leu Pro Asp Val Arg Asn Asn Phe

155 160 165

Tyr His Tyr Asn Gln Ser Asn Asp Ser Thr Ile Lys Leu Ile Ala

170 175 180

Met Leu Tyr Thr Pro Leu Arg Ala Asn Asn Ala Gly Asp Asp Val

185 190 195

Phe Thr Val Ser Cys Arg Val Leu Thr Arg Pro Ser Pro Asp Phe

200 205 210

Asp Phe Ile Phe Leu Val Pro Thr Val Glu Ser Arg Ile Leu

215 220 225

Asn Arg Ala Val Gly Arg Val Ala Asp Thr Ile Ala Ser Gly Pro

230 235 240

Val Asn Thr Glu Gln Ile Pro Ala Leu Thr Ala Val Glu Thr Gly

245 250 255

His Thr Ser Gln Val Val Pro Ser Asp Thr Met Gln Thr Arg His

260 265 270

Val Val Asn Tyr His Thr Arg Ser Glu Ser Ser Ile Glu Asn Phe

275 280 285

Met Gly Arg Ala Ala Cys Val Tyr Ile Ala Gln Tyr Ala Thr Glu

290 295 300

Lys Val Asn Asp Glu Leu Asp Arg Tyr Thr Asn Trp Glu Ile Thr

305 310 315

Thr Arg Gln Val Ala Gln Leu Arg Arg Lys Leu Glu Met Phe Thr

320 325 330

Tyr Met Arg Phe Asp Leu Glu Val Thr Phe Val Ile Thr Ser Ser

335 340 345

Gln Arg Thr Ser Thr Thr Tyr Ala Ser Asp Ser Pro Pro Leu Thr

350 355 360

His Gln Val Met Tyr Val Pro Pro Gly Gly Pro Ile Pro Arg Ser

365 370 375

Tyr Glu Asp Phe Ala Trp Gln Thr Ser Thr Asn Pro Ser Val Phe

380 385 390

Trp Thr Glu Gly Asn Ala Pro Pro Arg Met Ser Ile Pro Phe Met

395 400 405

Ser Val Gly Asn Ala Tyr Cys Asn Phe Tyr Asp Gly Trp Ser His

410 415 420

Phe Ser Gln Ser Gly Val Tyr Gly Tyr Thr Thr Leu Asn Asn Met

425 430 435

Gly His Leu Tyr Phe Arg His Val Asn Lys Ser Thr Ala Phe Pro

440 445 450

Val Asp Ser Val Ala Arg Val Tyr Phe Lys Pro Lys His Val Lys

455 460 465

Ala Trp Val Pro Arg Ala Pro Arg Leu Cys Pro Tyr Leu Tyr Ala

470 475 480

Gly Asn Val Asn Phe Asp Val Gln Gly Val Thr Glu Ser Arg Ser

485 490 495

Lys Ile Thr Leu Asp Arg Ser Thr His Asn Pro Leu Ser Asn Thr

500 505 510

His His His His His His His

515

<---

Claims (1)

A method for obtaining virus-like particles of norovirus containing the VP1 protein of enterovirus Echovirus 30 in vitro, including culturing E. coli cells transformed with a chimeric protein in a suitable culture medium, isolating and purifying virus-like particles, characterized in that a chimeric protein S _N -VP1 _E30 with a size of 516 is created amino acids with a molecular weight of 57 kDa, with a structure in accordance with the sequence of SEQ ID NO: 1, and E. coli cells, strain Rosette 2 DE3 are transformed with a plasmid containing a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1.

Images