Recombinant expression vector for producing virus-like particle-based norovirus multivalent vaccine and preparation method thereof

Abstract

The invention provides a fusion protein for enhancing soluble expression of a norovirus antigen and an expression vector of the fusion protein. More specifically, provided is a combination of a plurality of genetic norovirus antigen proteins and RID mutations, in which folding occurs most effectively when a recombinant fusion protein is formed in the case where a norovirus antigen is used as a target protein, the soluble expression yield of the prepared fusion protein is improved, and the assembly efficacy and homogeneity of a norovirus VLP can be enhanced. In addition, the present invention provides a method that can produce GII.4 and other various genetic types of Norovirus VLPs in Escherichia coli in large quantities and rapidly using the mutant RID, thereby enabling the development of VLP-based multivalent vaccines comprising more genetic types.

Description

Technical Field

[0001] This invention relates to recombinant expression vectors for the efficient and rapid production of norovirus multivalent vaccines based on virus-like particles in Escherichia coli and methods for preparing virus-like particles. More specifically, it relates to recombinant expression vectors capable of efficiently and solublely expressing antigen proteins of multiple genotypes of norovirus in Escherichia coli and efficient methods for preparing norovirus multivalent vaccines using the same.

[0002] This invention was completed with the support of the Ministry of Health and Welfare's Infectious Disease Prevention and Treatment Technology Development Project, "Development of a Multivalent Prophylactic Vaccine for Norovirus Enteritis VLP Type" (Project No.: 1465035890, Research Period: 2020.10.06~2023.12.31), and with the support of the Ministry of Education's Regional Base Vaccine R&BD Infrastructure Activation Project, "Development of Dosage Forms for Enhancing the Long-Term Stability of Norovirus VLP Type Vaccine Candidates" (Project No.: 2023112201, Research Period: 2021 / 07 / 01~2023 / 12 / 31). Background Technology

[0003] Norovirus, the virus to which this invention is applied, is one of the most important pathogens causing acute gastroenteritis and food poisoning. According to data released by the Foodborne Disease Burden Epidemiology Reference Group (FERG) under the WHO, more than 700 million norovirus infections are reported globally each year, with over 200 million cases in children under 5 years old, and 50,000 deaths annually. Norovirus infection is mainly transmitted through person-to-person contact, aerosols, the fecal-oral route, and contaminated water or food. Norovirus can infect people of all ages, but is more common in infants and the elderly. It can cause illness with very low viral loads, sheds the virus for a long time, and is stable to temperatures below 60 degrees Celsius. Furthermore, due to continuous antigenic variation and genetic recombination, it is difficult to acquire long-term, sustained immunity, leading to widespread outbreaks, such as in schools, hospitals, and nursing homes. The prominent symptoms caused by the pathogen are abdominal pain, low-grade fever, vomiting, and diarrhea. The typical incubation period is 24 to 48 hours, depending on the individual. Symptoms may appear asymptomatic, such as fever, vomiting, diarrhea, abdominal pain, and dehydration. In particular, the elderly may die from dehydration.

[0004] Norovirus is a non-enveloped virus belonging to the Caliciviridae family, with a diameter of approximately 30-40 nm. It consists of a 7.6 kbp single-stranded (+) RNA with three open reading frames (ORFs). ORF2 encodes VP1, the major structural protein that makes up the norovirus morphology, while ORF3 encodes VP2, which does not directly participate in structural formation. VP1 has a total size of approximately 59 kDa and forms dimers. Ninety dimers self-assemble, resulting in 180 VP1 units forming a single viral particle. The VP1 protein consists of two domains: the S domain, which plays a role in structural formation, and the P domain, which is known to be involved in inducing the actual immune response. The P2 site of the P domain binds to histo-blood group antigens (HBGAs), which are the target of vaccines.

[0005] Noroviruses are classified into seven genogroups based on the total amino acid sequence of VP1. GI, GII, and GIV induce human infection, with GII.4 being the most common genotype in outbreaks, accounting for 50-70% of the total prevalence. GII.4 alters antigenicity through mutations in the HBGA binding site of the P2 domain of VP1, allowing it to evade the immune system through various genetic recombinations, resulting in global epidemics with new variants emerging every two to three years. In addition to GII.4, a rapid epidemic of GII.17 occurred in Asia from 2014 to 2015 (Chan et al., Nature Communications 2015), and GII.3 has been frequently detected since 2016 (Kuang, et al. Gut Pathog 2019). Globally, the prevalence of GII.3 is increasing; in South Korea, the GII.3 / GII.2 genotype has been detected in the last three years. Norovirus is genetically highly diverse, with new variants constantly emerging. Therefore, the possibility of it becoming the dominant genotype, replacing GII.4, cannot be ruled out. Consequently, an effective vaccine for norovirus infection prevention requires the development of multivalent vaccines that are effective against as many genotypes as possible, including additional genotypes from the GII Genogroup, which accounts for over 90% of cases.

[0006] Traditionally, vaccines for viral infections are prepared from live vaccines that have been attenuated to reduce the virus's virulence, or inactivated vaccines obtained by purifying the virus in a culture medium. However, for norovirus, there is no known method for culturing the virus to replicate, making it difficult to develop live or inactivated vaccine formulations. Furthermore, suitable animal models have not been developed, resulting in the complete absence of currently commercially available norovirus vaccines. Therefore, vaccine development based on virus-like particles (VLPs) technology, which is not constrained by the ability of the virus to replicate in host cells, has become the mainstream approach.

[0007] Virus-like particles (VLPs) specifically express viral structural proteins, resulting in structures that resemble wild-type viruses in appearance. They are highly complex and intricate structures, ranging in size from 20 to 100 nm in diameter. Utilizing various recombinant protein expression systems, they specifically express known viral structural proteins, which can then be produced through virion assembly. While possessing a structure and morphology similar to actual infectious viruses and lacking viral genes, they cannot proliferate. However, they induce high immunogenicity through high-density and highly ordered conformation of antigens. Their structural similarity to wild-type viruses allows them to stimulate T-cell and B-cell immune pathways in vivo. However, their complex structure and the significant differences in characteristics between different viruses necessitate complex manufacturing processes. Due to factors such as low productivity, only a handful of VLPs have been approved for commercial use as vaccines.

[0008] Norovirus VLPs are primarily produced in baculovirus-insect cells, and are also known to be produced in yeast. However, these methods suffer from drawbacks such as long preparation times, the need for expensive production equipment, and high direct production costs. Considering that most deaths caused by norovirus infection occur in developing countries, there is a need for efficient and rapid production of low-cost vaccines. To achieve this, the production of E. coli-derived VLPs, which enables efficient and low-cost mass production of viral antigen proteins, is the preferred option. However, while E. coli can achieve efficient expression of the VP1 protein of various norovirus genotypes, it is well known that protein solubility is significantly reduced, resulting in low VLP formation efficiency. Therefore, to prepare E. coli-derived norovirus VLPs, it is first necessary to express a fusion protein with a soluble expression tag to improve the soluble expression rate of the antigen protein.

[0009] In previous studies, the inventors confirmed that when the RNA interaction domain (RID) containing the aminoacyl RNA synthetase N-terminal domain (ARSNTD) is fused to the N-terminus of the norovirus VP1 protein and expressed, not only is the soluble expression of the VP1 protein increased through the action of the intrinsically disordered peptide (IDP) structure of the RID, but also the intact VP1 antigen protein is prepared with high efficiency through a protein folding induction mechanism induced by tRNA attached to the RID. It has been verified that the target protein purified from the above fusion protein does not require an additional refolding process, and after purification, large quantities of virus-like particles (VLPs) can be rapidly produced through self-assembly (KR1914779 & KR 2120335).

[0010] However, it was observed that while the above invention worked well for the relatively stable G II.4 VP1 antigen protein, its soluble expression rate was significantly reduced when applied to other genotypes of norovirus VP1 protein. To address this issue, nine positively charged lysine (K) or arginine (R) residues in the full-length RID sequence were replaced with alanine (A). This resulted in a mutant (hereinafter, m9 RID) with lower tRNA binding efficiency but better suited to maintaining the amorphous structure (IDP). When applied, this mutant was more effective than the original unmutated RID (wild type RID, wtRID) for soluble expression of norovirus VP1 protein in various genotypes other than G II.4 (KR2038876).

[0011] In the case of m9 RID described above, soluble expression can be enhanced through IDP structure interaction, but tRNA binding is excluded. Therefore, the effect of inducing target protein folding is significantly reduced, resulting in high soluble expression levels but an increased proportion of soluble aggregates and a significantly reduced proportion of target proteins with the correct structure. Consequently, in the case of m9 RID, while soluble expression levels are high, the amount of VLP that can be obtained does not increase proportionally, thus resulting in low final preparation efficiency.

[0012] To address the aforementioned issues, the inventors performed protein motif analysis using databases such as Pfam, NCBI-CDD, and PROSITE, as well as the MOTIF tool. Based on the results, three or four lysine (K) or arginine (R) residues in the characteristic sequence of the original RID were replaced with alanine (A), thereby preparing novel mutant m3 RIDs or m4 RIDs that maintain the folding-induced function of target proteins based on binding to the original RNA and also maintain the IDP structure necessary for enhanced soluble expression.

[0013] When the above-mentioned mutant RID was attached to the N-terminus of the VP1 protein of various genotypes of norovirus and expressed, it was confirmed that not only was soluble expression significantly improved, but the efficiency of virus-like particle (VLP) preparation via self-assembly was also significantly improved, thus completing the present invention.

[0014] By utilizing the novel mutant RID (m3 or m4 RID) that is the core of this invention, which has the effect of inducing the folding of target proteins of RNA attached to the binding site by improving the soluble expression efficiency, a method is provided for the rapid and large-scale production of GII.4 and other genotypes of norovirus VLP in Escherichia coli, thereby verifying the inventiveness of the technology.

[0015] In particular, the significance of this invention lies in the ability to develop multivalent vaccines based on VLPs containing more genotypes, thereby providing appropriate solutions to address the threat of novel and acute epidemic pathogens with ongoing genetic variation. Summary of the Invention

[0016] Technical issues

[0017] The purpose of this invention is to provide a fusion protein and virus-like particles that can be used to prevent or treat infections caused by norovirus.

[0018] The main objective of this invention is to provide a mutant RID genetic combination in which a soluble fusion protein can be expressed most effectively when the norovirus antigen protein is used as the target protein.

[0019] Solution to the problem

[0020] To address the aforementioned technical problems, this invention provides a recombinant expression vector for producing norovirus vaccines, comprising a polynucleotide encoding a norovirus antigen protein; and a mutant protein in which three or four amino acid residues in the RNA interacting domain (RID) sequence isolated from mammalian-derived humanlysyl tRNA synthetase (hLysRS) are replaced with other amino acid residues.

[0021] In this invention, preferably, the RID consists of the amino acid sequences of SEQ ID NO. 2 and SEQ ID NO. 3.

[0022] Preferably, the substituted amino acid residues in the sequence of the RID of the present invention comprise at least one of the residues at positions 19, 23, 24, 30, 31, 38, and 40 of the amino acid sequence of SEQ ID NO. 3. More preferably, it may comprise at least one mutation of K19A, K23A, R24A, K30A, K31A, K38A, and K40A in the amino acid sequence of SEQ ID NO. 3. For example, amino acids at positions 23, 30, and 31 of the amino acid sequence of SEQ ID NO. 3 are substituted, or amino acids at positions 19, 38, and 40 are substituted. According to an embodiment of the present invention, the mutant protein of the RID may comprise one amino acid sequence from the amino acid sequences of SEQ ID NO. 5 to SEQ ID NO. 8.

[0023] Preferably, the norovirus antigen protein of the present invention can be an antigen protein whose genotype is not limited, but which contains at least one amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO. 13.

[0024] The present invention also provides a host cell transformed from the above-described expression vector. In this case, *Escherichia coli* (E. coli) is preferred.

[0025] According to another embodiment of the present invention, a fusion protein for a norovirus vaccine is provided, comprising a norovirus antigen protein and a mutant protein, wherein three or four amino acid residues in the sequence of the RNA interacting domain (RID) isolated from mammalian lysyl tRNA synthetase (hLysRS) are replaced with other amino acid residues. In the fusion protein of the present invention, at least one linker protein may be provided between the aforementioned norovirus antigen protein and the mutant RID protein.

[0026] The present invention also provides a virus-like particle, which is formed by the self-assembly of an antigen protein cleaved by RID in the above-mentioned fusion protein. The size of the above-mentioned virus-like particle (VLP) of the present invention can be from 30 nm to 40 nm.

[0027] According to another embodiment of the present invention, a method for producing a soluble norovirus antigen protein (or a method for producing norovirus-like particles) is provided, comprising: step a), producing a recombinant expression vector for producing a vaccine containing a polynucleotide, wherein the polynucleotide encodes a norovirus antigen protein and a mutant protein in which three or four amino acid residues in the sequence of the RNA-interacting domain (RID) isolated from mammalian lysyl tRNA synthetase are replaced with other amino acid residues; step b), introducing the expression vector into a host cell to produce a transformant; and step c), culturing the transformant and inducing the expression of the recombinant fusion protein to obtain the transformant.

[0028] In the above production method, the fusion protein may contain tobacco etch virus (TEV) protein between the norovirus antigen protein (the target protein of the present invention) and the RID protein. After step c), step d) may be included, using a TEV cleavage enzyme to cleave the RID protein and the norovirus antigen protein of the prepared fusion protein.

[0029] In addition, after step d) above, step e) may be included, which involves purifying the cleaved antigen protein; and after step e) above, a step of causing the purified protein to self-assemble into virus-like particles.

[0030] In the above-described production method of the present invention, the RID is composed of the amino acid sequences of SEQ ID NO. 2 and SEQ ID NO. 3, and the substituted amino acid residues may include at least one of the amino acid residues at positions 19, 23, 24, 30, 31, 38 and 40 of the amino acid sequence of SEQ ID NO. 3.

[0031] Most preferably, the mutant protein of the above-mentioned RID comprises one amino acid sequence from the amino acid sequences of SEQ ID NO. 5 to SEQ ID NO. 8.

[0032] In the above production method, the norovirus antigen protein may contain at least one amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO. 13.

[0033] The effects of the invention

[0034] The peptide of the present invention, which contains an amino acid sequence encoding a mutant RID protein, can significantly increase the soluble expression of norovirus antigen protein as a target protein when a recombinant protein (fusion protein) is produced in Escherichia coli.

[0035] Furthermore, this invention provides a mutant form of RID that, when using GII.4 and various genotype norovirus antigen proteins as target proteins to form recombinant proteins, folds most effectively and improves the final yield of virus-like particles.

[0036] Furthermore, this invention provides a method for the large-scale and rapid production of GII.4 and other genotypes of norovirus VLP in Escherichia coli using mutant RIDs, thereby enabling the development of multivalent VLP-based vaccines containing more genotypes. Attached Figure Description

[0037] Figure 1 is a schematic diagram of a recombinant expression vector for producing norovirus VP1 protein according to an embodiment of the present invention, and the expression results of highly efficient soluble protein based on expression level and whether or not the RID, as a fusion partner for soluble enhancement, is mutated. Figure 1a This is a schematic diagram illustrating the structure of a norovirus VP1 protein recombinant expression vector based on pET9a containing RID and mutant RID. Figure 1b To determine whether the soluble expression of the norovirus VP1 protein bound to RID was performed according to the genotype classification of norovirus, the results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1c Data were collected to compare the soluble expression levels of norovirus VP1 protein prepared according to an embodiment of the present invention or bound to mutant RID (mRID). Figure 1d The results were obtained by using the expression vector prepared according to the present invention and by SDS-PAGE to confirm whether RID and norovirus G II.17 VP1 protein bound to mutant RID were expressed efficiently or not. Figure 1e The results were obtained by transmission electron microscopy (TEM) to confirm the overall morphology of virus-like particles (VLPs) based on purified norovirus G II.17 VP1 protein expressed from four RID wild-type and mutant strains.

[0038] Figure 2aTo confirm the effect of the m3c mutation of RID on the soluble expression of norovirus GII.17VP1 fusion protein according to embodiments of the present invention, SDS-PAGE confirmed that, unlike norovirus GII.4VP1 protein, in the case of norovirus GII.17VP1 protein, if RID is fused to its N-terminus, it is mostly expressed in an insoluble state; conversely, if m3c is fused to its N-terminus, it is mostly expressed in a soluble state. Figure 2b The results of the nickel affinity chromatography purification of the norovirus GII.17 VP1 fusion protein containing m3c in the mutant RID, expressed by SDS-PAGE according to embodiments of the present invention, were obtained by confirming the protein separation. Figure 2c The results of the series of processes from the initial TEV enzyme cleavage of the RID (EE domain+m3c)-norovirus GII.17 VP1 fusion protein eluted by nickel affinity chromatography to the fractionation of the FT (flow through) eluted by nickel affinity chromatography were confirmed by SDS-PAGE. Figure 2d The results were obtained by SDS-PAGE to confirm the purification status of norovirus GII.17 VP1 protein eluted from nickel affinity chromatography by ion exchange resin replacement chromatography. Figure 2e The results show the diameter distribution of virus-like particles (VLPs) prepared after the self-assembly step of purified norovirus GII.17 VP1 protein, analyzed by dynamic light scattering (DLS). Figure 2f Results of confirming the overall morphology of the prepared norovirus GII.17 virus-like particles by transmission electron microscopy (TEM). Figure 2g This is a schematic diagram illustrating a binding configuration for HBGA binding affinity analysis according to an embodiment of the present invention. Figure 2h To analyze the degree of binding of GII.4, GII.17, and GII.3 VLP proteins to Blood type B (tri), Blood type B (tri), and Le a (H type 1), respectively, the corresponding binding affinity of each VLP was labeled using EC50.

[0039] Figure 3a This is a schematic diagram illustrating the experimental procedure performed to determine the specific antibody-inducing ability of GII.17 virus-like particles (VLPs) prepared according to an embodiment of the present invention. Figure 3ba , Figure 3bb and Figure 3bc The results show the degree of specific antibody induction in serum samples collected after one, two, and three administrations of the virus-like particles (NoVGII.17 VLP) of the present invention, determined by enzyme-linked immunosorbent assay (ELISA). Figure 3c To demonstrate the effectiveness of the above-mentioned immune serum in analyzing the GII.17 VLP antigen (a VLP composed of antigen protein purified after cleavage of the RID (EE domain+m3c)-NoVGII.17 VP1 fusion protein of the present invention), Figure 3c A schematic diagram of the binding configuration for the binding inhibitory effect between Nov VLP and HBGA. Figure 3d To confirm the differences in HBGA binding inhibition function among the 1-, 2-, and 3-dose groups after each dose of norovirus GII.17VLP in an embodiment of the present invention.

[0040] Figure 4a To confirm the effect of RID mutation on the soluble expression of norovirus GII.3 VP1 protein according to embodiments of the present invention, SDS-PAGE confirmed that proteins with RID fused to the N-terminus mostly exhibited an insoluble expression state, while those with RID (EE domain+m3c) fused to the N-terminus were mostly expressed as soluble proteins. Figure 4b The results of the nickel affinity chromatography purification of norovirus GII.3 VP1 protein containing m3c in mutant RID, expressed by SDS-PAGE, were used to confirm the protein separation during the purification process according to embodiments of the present invention. Figure 4c This is the result of a series of processes from the initial cleavage of the RID (EE domain+m3c)-norovirus GII.3 VP1 fusion protein eluted by nickel affinity chromatography to the fractionation of the FT (flow through) eluted by nickel affinity chromatography, using TEV enzyme cleavage. Figure 4d The results were obtained by SDS-PAGE to confirm the purification status of norovirus GII.3 VP1 protein eluted from nickel affinity chromatography by ion exchange resin replacement chromatography. Figure 4e The results show the diameter distribution of virus-like particles (VLPs) prepared by dynamic light scattering (DLS) after the self-assembly step using purified norovirus GII.3 protein. Figure 4fResults of confirming the overall morphology of the prepared norovirus GII.3 virus-like particles by transmission electron microscopy (TEM).

[0041] Figure 5a This is a schematic diagram illustrating the experimental procedure performed to determine the specific antibody-inducing ability of GII.3 virus-like particles (VLPs) prepared according to an embodiment of the present invention. Figure 5ba , Figure 5bb and Figure 5bc The results show the degree of specific antibody induction in serum samples collected after one, two, and three applications of the virus-like particles (NoVGII.3 VLP) of the present invention, determined by enzyme-linked immunosorbent assay (ELISA). Figure 5c To confirm the differences in HBGA binding inhibition function among the 1-, 2-, and 3-dose groups after each dose of norovirus GII.3 VLP in an embodiment of the present invention.

[0042] Figure 6a To confirm the effect of expression levels and the presence or absence of m3c mutations in RID, a fusion partner for enhanced solubility, on the soluble expression of proteins fused with norovirus GI.1 VP1 protein, SDS-PAGE analysis revealed that proteins fused with RID at the N-terminus were predominantly insoluble, while those fused with RID (EE domain + m3c) at the N-terminus were predominantly soluble. Figure 6b To confirm the effect of mutations in the RID (especially the SE domain in the RID) on the soluble expression of norovirus GI.3 VP1 fusion protein according to embodiments of the present invention, SDS-PAGE confirmed that proteins with RID fused to the N-terminus were mostly expressed in an insoluble state, while those with RID (EE domain + m3c) fused to the N-terminus were mostly expressed as soluble proteins. Figure 6c According to embodiments of the present invention, the effect of the m3c mutation of the norovirus GII.2 VP1 protein and its target protein on the soluble expression of a fusion protein with or without the RID as a soluble enhancement fusion partner, based on expression levels, was confirmed. SDS-PAGE confirmed that, in the case of a protein with the RID fused to its N-terminus, the majority of the expression was insoluble; conversely, if the RID (EE domain + m3c) was fused to and expressed at its N-terminus, the majority of the expression was soluble.

[0043] Figure 6daThe size distribution of norovirus GI.1 VLP particles prepared solely from the norovirus GI.1 VP1 protein ultimately prepared by expressing the fusion protein with RID (EE domain+m3c) was confirmed by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. Figure 6da (Upper end) and the results of particle formation. Figure 5db shows the size distribution of norovirus GI.1 VLP particles prepared solely from the norovirus GI.3 VP1 protein finally purified after expression of the fusion protein with RID (EE domain+m3c) according to the present invention. Figure 6db The diagram shows the formation of particles (at the top). Figure 6dc To confirm the size distribution of norovirus GII.2 VLP particles prepared solely from the norovirus GII.2 VP1 protein finally purified after expression of the fusion protein with RID (EE domain+m3c) according to the present invention ( Figure 6dc (The upper part) and the diagram of particle formation. Detailed Implementation

[0044] The preferred embodiments of the present invention are described in detail below. The advantages and features of the present invention will become clear from the following embodiments in which they are implemented. However, the present invention is not limited to the embodiments disclosed below, and can be implemented in various different forms. These embodiments are provided to complete the disclosure of the present invention and to fully inform those skilled in the art to which this invention pertains. The present invention is defined only by the scope of the claims.

[0045] Throughout this specification, the same reference numerals refer to the same constituent elements. Unless otherwise defined, all terms used herein (including technical and scientific terms) will be understood in a meaning commonly understood by one of ordinary skill in the art to which this invention pertains. Furthermore, unless explicitly defined, terms as defined in commonly used dictionaries will not be interpreted unreasonably or excessively. The terminology used herein is for illustrative purposes and not for limiting the invention. In this document, the singular form includes the plural form unless specifically stated otherwise.

[0046] The term "target protein" as used in this specification refers to a protein that a person skilled in the art would like to produce in large quantities, which can be expressed in a host cell by inserting a polynucleotide encoding the target protein into the recombinant expression vector of the present invention.

[0047] According to one embodiment of the present invention, the target protein may include viral antigen proteins, antibodies, cell receptors, enzymes, structural proteins, serum, and cellular proteins. According to a preferred embodiment of the present invention, the viral antigen protein is a norovirus antigen protein.

[0048] The norovirus antigen protein of the present invention may include GI and GII of norovirus types, and more preferably, may include norovirus GI.1, GI.3, GII.17, GII.2, and GII.3 VP1 antigen proteins. The norovirus VP1 antigen protein of the present invention may be composed of the amino acid sequences represented by SEQ ID NO. 9 to SEQ ID NO. 13, but is not limited thereto.

[0049] As used in this specification, the terms “RID,” “RNA interacting domain,” or “N-terminal appended RNA binding domain of Lysyl tRNA synthetase (ARSNTD)” refer to the intrinsic N-terminal extension site of mammalian LysRS, which is involved in the interaction between RNA and other proteins.

[0050] According to one embodiment of the present invention, the RID protein described above can be used as a fusion partner to increase the expression level or solubility (or water solubility) of the target protein described above.

[0051] According to one embodiment of the present invention, the above-mentioned RID is the amino acid sequence (wild type) of the N-terminal appended RNA interacting domain (hRID) isolated from human lysyl tRNA synthetase (hLysRS), and the mutant RID contains a mutant sequence in which one or more positively charged amino acid residues in the above-mentioned RID sequence are replaced by other amino acid residues.

[0052] According to a preferred embodiment of the present invention, the RID protein of the present invention comprises an EE domain (SEQ ID NO. 2, MSAVKAA) used to increase the expression level of the target protein and an SE domain (SEQ ID NO. 3) used to enhance soluble expression by inducing folding of the target protein.

[0053] The RID protein used in this invention can be mutated. As used in this specification, the term "mutation" refers to the substitution of one or more residues in an amino acid sequence by other amino acid residues, the deletion of some residues in an amino acid sequence, or the insertion of a specific amino acid residue. In this invention, "mutation" is used interchangeably with "point mutation" or "site-directed mutation," referring to a mutation in which an amino acid residue at a specific position is substituted by another amino acid residue.

[0054] In this specification, the amino acid sequence of “RID” may include the amino acid sequences of SEQ ID NO. 2 and SEQ ID NO. 3.

[0055] According to one embodiment of the present invention, the mutation of RID preferably involves the substitution of 3 to 4 amino acid residues in the SE domain. For example, in the amino acid sequence of SEQ ID NO. 3 (in this invention, the SE domain in RID), 3 to 4 amino acid residues may be substituted. According to a preferred embodiment of the present invention, in the above-mentioned amino acid sequence of the SE domain (SEQ ID NO. 3), the substituted amino acid residues may include at least one of positions 19, 23, 24, 30, 31, 38, and lower 40. For example, in the amino acid sequence of SEQ ID NO. 3, amino acids 23, 30, and 31 are substituted, or amino acids 19, 38, and 40 may be substituted. The substituted amino acids preferably include at least one of K19A, K23A, R24A, K30A, K31A, K38A, and K40A. More preferably, in the amino acid sequence of the RID SE domain (SEQ ID NO. 3), one amino acid residue at positions 23, 24, 30, and 31 (e.g., K23, R24, K30, and K31) may be substituted. That is, according to an embodiment of the present invention, the amino acid sequence of the mutated RID according to the present invention may include, in addition to SEQ ID NO. 2, one of the sequences from SEQ ID NO. 5 to SEQ ID NO. 8.

[0056] The names of the sequences used in this invention are organized as shown in Table 1 below.

[0057] [Table 1]

[0058]

[0059] In this invention, the EE domain may contain the amino acid sequence of SEQ ID NO. 2 or a portion thereof.

[0060] According to one embodiment of the present invention, when producing the fusion protein of the present invention, with norovirus antigen protein as the target protein, compared to the unmutated RID protein, the fusion protein exhibits higher solubility (or water solubility) when binding the mutated RID protein (a protein composed of one amino acid sequence from SEQ ID NO. 4 to SEQ ID NO. 8 and the amino acid sequence of SEQ ID NO. 2) (see reference). Figure 1c Furthermore, in RID, compared to the m9 protein with 9 mutations in the SE domain, the m3 or m4 proteins, which have substituted 3 or 4 amino acid residues, exhibit better protein folding when bound to the norovirus antigen protein (see [reference]). Figure 1e In the SE domain sequence, when RID containing m3 or m4 substituted with 3 or 4 amino acid residues is bound to norovirus antigen protein, it shows an enhanced soluble expression yield and improves VLP formation efficiency by inducing more accurate folding.

[0061] As used in this specification, the term "expression vector" refers to a linear or circular DNA molecule composed of fragments encoding a target protein operatively linked to an additional segment provided for transcription into the expression vector. This additional segment, as described above, includes a promoter and a stop codon sequence. Expression vectors may also include one or more origins of replication, one or more selection markers, etc. Expression vectors are typically induced by plasmid or viral DNA, or elements of both. The term "operatively linked" as used in this specification refers to the arrangement of fragments to function as a means of initiating transcription in a promoter and via a coding sequence, using a stop codon.

[0062] In the expression vector according to the present invention, the expression vector may be a plasmid, a viral vector, a bacteriophage particle, or a genomic insert. The expression vector is transformed into a host cell and can then replicate or integrate into the host cell's genome, independent of the host cell's genome.

[0063] According to one embodiment of the present invention, the above-mentioned polynucleotide may also encode a "linker", and at least one linker protein may be respectively set between the target protein and the RNA interacting domain (RID) protein.

[0064] According to a preferred embodiment of the present invention, the fusion protein sequence of the present invention, in particular, may contain a TEV-specific cleavage sequence (TEV-specific cleavage site) for cleaving RID within the sequence of the target protein.

[0065] In the recombinant expression vector for vaccine production of the present invention, the protein cleavage enzyme can be TEV.

[0066] The present invention also provides a host cell transformed from the above expression vector.

[0067] As used in this specification, the terms "transformation" or "introduction" refer to the introduction of DNA into a host, enabling the DNA to replicate as an extrachromosomal factor or through chromosomal integration. The expression vector transformation methods of this invention may include, but are not limited to, electroporation, calcium phosphate (CaPO4) method, calcium chloride (CaCl2) method, microinjection method, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, or lithium acetate-DMSO method.

[0068] The present invention also provides a fusion protein comprising a target protein and a RID protein. In the present invention, the aforementioned RNA interacting domain (RID) protein may contain mutant RIDs with three (m3) to four (m4) amino acid residues substituted in the SE domain.

[0069] As used in this specification, the term "fusion protein" refers to a protein in which other proteins or additional amino acid sequences are attached to the N-terminus or C-terminus of the target protein sequence. Furthermore, in this invention, the aforementioned fusion protein can be a vaccine produced using the recombinant expression vector of this invention. According to one embodiment of the invention, the fusion protein of this invention is a protein formed by fusing a mutant RID with a target protein (norovirus antigen protein).

[0070] According to one embodiment of the present invention, the above-mentioned recombinant expression vector can be used to form a fusion protein, which can be used as a vaccine by itself or by any additional process.

[0071] The present invention also provides a virus-like particle (VLP) composed of multiple antigen proteins cleaved by the RID in the above-mentioned fusion protein. The virus-like particle of the present invention cleaves the mutant RID of the above-mentioned fusion protein during its formation process to form a purified antigen protein (e.g., norovirus VP1 antigen protein).

[0072] The present invention will now be described in more detail through examples.

[0073] These embodiments are merely illustrative of the invention, and the scope of the invention should not be construed as being limited to these embodiments, as will be apparent to those skilled in the art.

[0074] [Example 1]

[0075] Preparation of norovirus fusion protein containing mutant RID

[0076] <Example 1-1>

[0077] To prepare virus-like particles composed of norovirus GII.17 VP1 protein, the VP1 gene (Norovirus Hu / GII.17 / HKG / 2014 / CUHK-NS-405, NCBI access number: KP902566.1), a source of norovirus GII.17 protein, was utilized. The norovirus antigen VP1 consists of 539 amino acids, with a protein molecular weight of approximately 59.2 kDa. Based on this protein sequence, a gene sequence (SEQ ID NO. 9) was synthesized in *E. coli* after codon optimization. (The gene was synthesized by Bionics, Japan.)

[0078] In this embodiment, the RID EE domain peptide (MSAVKAA (SEQ ID NO. 2)) and the RID SE domain peptide (SEQ ID NO. 3), which have the effects of increasing expression and solubility, were used at the N-terminus of the norovirus GII.17 VP1 protein. Furthermore, to confirm the effect of RID mutation, instead of the aforementioned RID SE domain peptide, mutant RID SE domain peptides with more than 50% similarity to SEQ ID NO. 3 were used, including: mutant RID SE domain peptides containing mutations of K19A, K38A, and K40A (SEQ ID NO. 5), mutant RID SE domain peptides containing mutations of K23A, K30A, and K31A (SEQ ID NO. 7), or nine mutant RID SE domain peptides (SEQ ID NO. 4). In this embodiment, a recombinant protein fused with each mutant SE domain was prepared, and the soluble expression state was verified.

[0079] Furthermore, the known ENLYFQG / S sequence serves as the TEV cleavage site. To achieve more efficient cleavage, a peptide with the amino acid sequence (GaSb)n (a≥1, b≥1, n≥1) was devised and placed between the TEV cleavage sequence (ENLYFQ) and the aforementioned target protein (norovirus antigen protein) sequence. A schematic diagram of the fusion protein expression vector prepared in this embodiment is shown below. Figure 1a .

[0080] The gene containing the above sequence was inserted using the cleavage enzymes NdeI and BamHI present in the MCS of the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector, and the sequence of the final recombinant expression vector is as follows.

[0081] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GII.17 VP1

[0082] [(SEQ ID NO. 2)-(SEQ ID NO. 3)-GT-6xHIS-TEV-GS-(SEQ ID NO. 9)]

[0083] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0084] Name the complex prepared above as "RID-NoV GII.17 VP1".

[0085] MSAVKAA-m3a-GT-6xHIS-TEV-GS-NoV GII.17 VP1

[0086] [(SEQ ID NO. 2)-(SEQ ID NO. 5)-GT-6xHIS-TEV-GS-(SEQ ID NO. 9)]

[0087] :MSAVKAA-AAVQAAEVKVDGSEPKLSANELKRRLKAEKKVAEKEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0088] Name the complex prepared above as "RID(EE domain+m3a)-NoV GII.17 VP1".

[0089] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GII.17 VP1

[0090] [(SEQ ID NO. 2)-(SEQ ID NO. 7)-GT-6xHIS-TEV-GS-(SEQ ID NO. 9)]

[0091] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0092] Name the complex prepared above as "RID(EE domain+m3c)-NoV GII.17 VP1".

[0093] MSAVKAA-m9 RID-GT-6xHIS-TEV-GS-NoV GII.17 VP1

[0094] [(SEQ ID NO. 2)-(SEQ ID NO. 4)-GT-6xHIS-TEV-GS-SEQ ID NO. 9)]

[0095] :MSAVKAA-AAVQAAEVKVDGSEPKLSANELAARLAAEAAVAEAEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV

[0096] -GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0097] Name the complex prepared above as "RID (EE domain + m9)-NoV GII.17 VP1".

[0098] <Example 1-2>

[0099] The expression vector prepared in Example 1-1 above was transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed E. coli were cultured in 3 ml of LB medium containing 50 μg / ml kanamycin at 37°C and 250 rpm for 5 to 7 hours. Then, 1 ml was transferred to 10 ml, diluted 10-fold, and cultured for further. After about 2 to 4 hours, when the O.D600 reached 0.5 to 0.7, 0.4 mM isopropyl-β-D-thiogalactoside (IPTG, Biosang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17 to 19 hours (maximum 21 hours). The culture medium was centrifuged to obtain only the precipitated *E. coli*, which was then cryopreserved at -80°C. For each 3 ml volume of the harvested *E. coli*, 0.3 ml of a dissolution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added. After ultrasonic pulverization, the total lysate (T) was separated into precipitate (P) and soluble lysate (S) by centrifugation. The results were analyzed by SDS-PAGE. Figure 1d As shown, norovirus GII.17 VP1 protein with RID added to the N-terminus is efficiently expressed, but mostly insoluble. In contrast, norovirus GII.17 VP1 protein with mutant RID added to the N-terminus is shown to be efficiently soluble.

[0100] <Examples 1-3>

[0101] Using the same culture method as in Examples 1-2 above, which confirmed efficient soluble expression, cells obtained by inducing expression in 500 ml of *E. coli* culture were resuspended in 75 ml of dissolution buffer A [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole]. The cell suspension was then disrupted using an ultrasonic cell disruptor. At 4°C, the lysed cells were centrifuged at 13500 rpm for 10 minutes. The supernatant was collected and Ni+ affinity chromatography was performed using AKTA (GE Healthcare). First, the Ni Sepharose High Performance (Cytiva) column was equilibrated with buffer A (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole). The cell lysate supernatant was then loaded into the equilibrated column at a flow rate of 3-5 ml / min. Buffer A and buffer B (50 mM Tris-HCl (pH 7.5), containing 300 mM imidazole and 50 mM Tris-HCl (pH 7.5) were then used. Norovirus GII.17 VP1 protein was obtained by eluting and injecting imidazole in a linear concentration gradient from 10 mM to 300 mM using 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole.

[0102] The separation status of the target protein was confirmed by SDS-PAGE, and the gel was analyzed using Image Lab (Ver. 6.1). Using the program's Quantity Tool, the Total Lysate Band was designated as the Reference Band, and the relative quantity of the supernatant was used to compare and analyze the expression levels of soluble proteins in each construct. The results are as follows: Figure 1c As shown, it can be confirmed that the soluble protein expression rate and purified protein yield of the fusion protein containing m3c in the RID with 3 mutations are better than those of the case containing m3a.

[0103] <Examples 1-4>

[0104] VLP formation was confirmed based on the type of fusion protein that binds to the norovirus antigen protein.

[0105] The morphology of virus-like particles (VLPs) composed of purified norovirus GII.17 VP1 protein was observed using electron microscopy. The VLPs composed of norovirus VP1 were first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, and dried at room temperature for 10 minutes. The formation of the purified norovirus VP1 protein VLPs was then confirmed using transmission electron microscopy (TEM; Talos L120C, FEI, Czech Republic). Figure 1e As shown, it was confirmed that the diameter of the identified VLPs formed by norovirus particles was between 30 and 40 nm, resulting in spherical VLPs. In particular, the norovirus GII.17 VP1 protein fused with the mutant RID containing m3c formed homogeneous VLPs of almost similar size with high efficiency. Conversely, in the case of other mutant RIDs, VLP formation was observed, but with low efficiency and containing many intermediates and impurities.

[0106] The results of Example 1 above confirm that when the norovirus VP1 antigen protein of the present invention containing a mutant RID with preserved RNA binding sites is expressed, not only can a higher level of expression be achieved, but also appropriate protein folding is induced, thereby showing higher solubility, and the efficiency and homogeneity of VLP assembly of the finally purified VP1 protein are excellent.

[0107] [Example 2]

[0108] Preparation of norovirus GII.17 VP1 virus-like particles (VLPs) with N-terminal expression levels and soluble enhancing partners (RIDs).

[0109] <Example 2-1>

[0110] The two expression vectors (RID-NoV GII.17 VP1 expression vector and RID(EEdomain+m3c)-NoV GII.17 VP1 expression vector) from Examples 1-1 above were transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed E. coli were cultured in 3 ml of LB medium containing 50 μg / ml kanamycin at 37°C and 250 rpm for 5 to 7 hours. After that, 1 ml was transferred to 10 ml, diluted 10-fold, and cultured for further. Approximately 2 to 4 hours later, when the O.D600 reached 0.5–0.7, 0.4 mM of isopropyl-β-D-thiogalactoside (IPTG, Biosang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the culture was incubated at 16°C for 17 to 19 hours (maximum 21 hours). The culture medium was centrifuged to obtain only the precipitated E. coli, which was then cryopreserved at -80°C. 0.3 ml of dissolution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added to 3 ml of E. coli harvested culture medium. After ultrasonic pulverization, the total lysate (T) was separated into precipitate (P) and supernatant (S) by centrifugation. SDS-PAGE analysis confirmed the separation of the lysate. Figure 2a As shown, norovirus GII.17 VP1 protein with mutant RID added to the N-terminus exhibits highly efficient soluble expression.

[0111] <Example 2-2>

[0112] Using the same culture method as in Examples 1-2 above, which confirmed efficient soluble expression, cells obtained by inducing expression in 500 ml of *E. coli* culture were resuspended in 75 ml of dissolution buffer A [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole]. The cell suspension was then disrupted using an ultrasonic cell disruptor. At 4°C, the lysed cells were centrifuged at 13500 rpm for 10 minutes. The supernatant was collected and Ni+ affinity chromatography was performed using AKTA (GE Healthcare). First, the Ni Sepharose High Performance (Cytiva) column was equilibrated with buffer A (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole). The cell lysate supernatant was then loaded into the equilibration column at a flow rate of 3-5 ml / min. Buffer A and buffer B (50 mM Tris-HCl (pH 7.5), containing 300 mM imidazole and 50 mM Tris-HCl (pH 7.5) were then used. Norovirus GII.17 VP1 protein was eluted and obtained by injecting imidazole in a linear concentration gradient from 10 mM to 300 mM using 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole.

[0113] The separation status of the target protein was confirmed by SDS-PAGE of the above eluents. The corresponding fractionated eluents were dialyzed overnight (12 to 16 hours) at 4°C in dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol]. The N-terminal RID and norovirus antigen protein VP1 were cleaved using TEV cleavage enzyme.

[0114] To separate and purify the removed target proteins, such as RID, which are N-terminal fusion partners, a Ni Sepharose High Performance (Cytiva) column was used. The column was equilibrated with the same solution A as the dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% 2-mercaptoethanol] and loaded into the column at a flow rate of 3–5 ml / min. The removed NoV GII.17 target proteins, such as RID, which are N-terminal fusion partners, were recovered in a form that was not adsorbed onto the column and eluted.

[0115] To increase the final purity of the target protein, ion exchange chromatography was performed using AKTA (GE Healthcare). The column (Fractogel EMD DEAE(M), Merck Millipore) was equilibrated with buffer solution A [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol)]. After loading, the column was passed through buffer solutions AA and BB [50 mM Tris-HCl (pH 8.5), 1 M NaCl, 5% glycerol, 0.1% β-mercaptoethanol)] at a flow rate of 1 ml / min. The target protein was eluted by injecting NaCl with a linear concentration gradient from 10 mM to 1 M, and each solution was fractionated to obtain the final purity. The purification status and purity are as follows: Figure 2b , Figure 2c , Figure 2d As described in the document (see Figure 2), the final purified protein concentration was quantified using BSA (Amresco, Soren, Ohio, USA). As shown in Figure 2, the RID protein bound to the fusion protein was isolated, thus effectively purifying the target protein.

[0116] <Example 2-3>

[0117] The norovirus VP1 protein purified according to Example 2-2 was in a state containing 10% glycerol. The final state of the assembly buffer [100mM potassium phosphate, pH 5.8, 150mM NaCl, 10% glycerol] was changed overnight (12 to 16 hours) and stored at 4°C. The purified norovirus VP1 protein was assembled in VLP form through a series of processes.

[0118] <Example 2-4>

[0119] To confirm the overall diameter distribution of virus-like particles (VLPs) assembled with the purified norovirus VP1 protein according to Examples 2-3, dynamic light scattering (DLS) analysis was performed. In the analysis of the above complex, 1 mL of sample was added to a cuvette at 16°C using DLS (Particular Systems, Zetasizer Nano Family) and the measurement was performed. In the dynamic light scattering analysis of cells, the total intensity and mass of each region were used to confirm the distribution in the image. Figure 2e It was confirmed that particles with a diameter of 30–40 nm were formed within the hydrodynamic radius of the light source. (DLS values ​​can be affected by variations in light wavelength.)

[0120] <Examples 2-5>

[0121] The morphology of virus-like particles (VLPs) composed of purified norovirus GII.17 protein was observed using an electron microscope. The purified VLPs were first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, dried at room temperature for 10 minutes, and then photographed using a transmission electron microscope (TEM; Talos L120C, FEI, Czech Republic). Figure 2f As shown, the purified and assembled norovirus VP1 protein formed a VLP. It was confirmed that the diameter of the identified VLP was between 30 and 40 nm, the diameter of the norovirus particle, and that highly homogeneous spherical VLPs could be formed (see reference). Figure 2f ).

[0122] <Example 2-6>

[0123] To determine the degree of binding of VLPs of GII.4, GII.17, and GII.3 purified according to the present invention to HBGA, an HBGA binding assay was performed. Figure 2g As shown, five biotin-coated HBGAs (H (type 2)-PAA-biotin (01-034), Lea-PAA-biotin (01-035), Led (H type 1)-PAA-biotin (01-037), Blood type A (tri)-PAA-biotin (01-032), Blood type B (tri)-PAA-biotin (01-033) (Glycotech)) were added to a 96-well plate (High Binding Capacity (HBC) NeutrAvidin plates (Thermo Scientific™)) coded with streptavidin at a concentration of 10 μg / ml, 100 μl per well, and reacted at room temperature for 5 hours. After washing the plate with PBST, GII.17 was added. VLP protein was diluted in PBS, 100 μL per well, and incubated at 4°C for 18 hours. After washing the plate with PBST, rabbit NoV GII.17 polyclonal IgG Ab was diluted 2000-fold in PBS, 100 μL per well, and incubated at room temperature for 1 hour and 30 minutes. After washing with PBST, goat-derived rabbit IgG (Goat-anti rabbit IgG HRP) was diluted 10000-fold in PBS, 100 μL per well, and incubated at room temperature for 1 hour and 30 minutes. Afterwards, the plate was developed with TMB solution at room temperature for 5 minutes, and the reaction was stopped with 0.5 M sulfuric acid solution. The absorbance was measured at 450 nm. The binding between norovirus VLP and five HBGAs was confirmed. The results confirmed that GII.4, GII.17, and GII.3 VLP proteins strongly bind to Blood type B (tri), Blood type B (tri), and Le a (H type 1), respectively. The degree of binding was labeled by EC50, which is consistent with... Figure 2h same.

[0124] [Example 3]

[0125] Immunogenicity of the formed GII.17 VLP was confirmed using mRID.

[0126] <Example 3-1>

[0127] To determine the ability of norovirus GII.17 antigen-specific antibodies to induce antibodies, virus-like particles (VLPs) prepared according to the present invention (VLPs composed of antigen proteins purified after cleavage of the RID in the fusion protein (RID(EE domain +m3c)-NoV GII.17 VP1) of the present invention) were injected intramuscularly into a group of 7-week-old Sprague Dawley (SD) rats (5 rats / group) at a concentration of 500 μL. Figure 3a Using a syringe fitted with a 26-gauge needle, the prescribed dosage (500 μL / head) was injected into both thighs of the animal, mixed with norovirus GII.17 VLP antigen and 0.5 mg of the immunostimulant aluminum hydroxide. The vaccine was administered three times, every two weeks. Approximately 1 mL of whole blood was collected via the jugular vein on the scheduled date and injected into a vacuum blood collection tube containing a clot activator.

[0128] To determine the target protein-specific antibody titers in serum obtained from each animal experimental group, an enzyme-linked immunosorbent assay (ELISA) was performed. Norovirus GII.17 VLP was added to 96-well plates (High Binding Capacity (HBC) NeutrAvidin plates, Thermo Scientific™) at a concentration of 0.5 μg / mL, 100 μL per well. After coating overnight at 4°C, 200 μL of 3% BSA solution was added, and the plates were incubated at 37°C for 2 hours and then blocked. After washing the plates with PBST, the immune serum was diluted (1:1600 dilution), 100 μL per well, and incubated at 37°C for 1 hour and 30 minutes. After washing the plates with PBST, to detect antibodies bound to GII.17 VLP in the serum, antibodies against goat-derived rat IgG (Southern Biotech 1030-05, Goat-anti-mouse IgG HRP) were administered at 37°C for 1 hour. Subsequently, TMB (sigma) was used to detect antibodies. After the T0440 solution was incubated at room temperature for 5 minutes, the colorimetric reaction was stopped with 0.5M sulfuric acid solution, and the absorbance was measured at 450 nm. The results of the GII.17 VLP-specific antibody titers in blood samples collected after vaccination at 8000-fold dilutions (1 μg / head, 5 μg / head, 10 μg / head, and 20 μg / head) confirmed the positive results. Figures 3ba to 3bcAs shown, only two doses were required to generate comparable GII.17 VLP antigen-specific antibodies at concentrations of 5 μg, 10 μg, and 20 μg. Specifically, in the 0.2 μg / head test group, antibody production was confirmed in all individuals 14 days after the second administration.

[0129] <Example 3-2>

[0130] To determine the immunosuppressive effect of GII.17 VLP antigen (composed of the antigen protein purified after RID cleavage from the RID(EE domain+m3c)-NoVGII.17VP1 fusion protein of this invention), Figure 3c The degree of binding between Nov VLP and HBGA was determined, and an HBGA blocking assay was performed on the obtained immune serum according to the schedule after vaccination. Figure 3c In 96-well plates coated with streptavidin (High Binding Capacity (HBC) NeutrAvidin plates (Thermo Scientific™), USA), 10 μg / ml of Blood type B(tri)-PAA-biotin (01-033) (Glycotech), which has high binding affinity for GII.17, was added to each well, and the reaction was carried out at room temperature for 5 hours. First, second, and third immune sera, used to confirm the inhibitory efficacy of antigen-HBGA binding, were sequentially diluted 100-fold to 2-fold in PBS and reacted with appropriately diluted GII.17 VLP protein at 37°C for 1 hour and 30 minutes. The serum and GII.17... VLP reaction solution was added at 100 μL per well to the plate reacting with HBGA, and incubated at 4°C for 18 hours. After washing the plate with PBST, rabbit NoVGII.17 polyclonal IgG Ab was diluted 2000-fold with PBS, and 100 μL was added to each well, and incubated at room temperature for 1 hour and 30 minutes. After washing with PBST, goat-derived rabbit IgG (Goat-anti rabbit IgG HRP) was diluted 30000-fold with PBS, and 100 μL was added to each well, and incubated at room temperature for 1 hour and 30 minutes. Afterward, the reaction was developed with TMB solution at room temperature for 5 minutes, and then stopped with 0.5 M sulfuric acid solution. The absorbance was measured at 450 nm.

[0131] Figure 3dThe results of the HBGA binding inhibition assay in immune serum (using B tri to synthesize carbohydrates) were used to confirm the HBGA inhibitory efficacy at 5 μg / head (low concentration), 10 μg / head (medium concentration), and 20 μg / head (high concentration) 14 days after 1, 2, and 3 vaccinations. Figure 3d It was confirmed that the inhibitory efficacy of HBGA binding increased dose-dependently from the first immunization serum. When analyzing the GII.17 VLP results, an HBGA binding inhibition capacity of ≥1:200 was confirmed at all doses after the first vaccination, a high HBGA binding inhibition capacity of ≥1:5000 was confirmed at all doses after the second vaccination, and a high HBGA binding inhibition capacity of ≥1:6000 was confirmed at all doses after the third vaccination. The degree of inhibition of VLP antigen-HBGA binding was not significant at all concentrations. The degree of inhibition of VLP antigen-HBGA binding at all concentrations was higher after the second and third immunization sera compared to the first vaccination. The inhibitory efficacy at each application concentration differed greatly and saturated between the low and medium-high concentration groups.

[0132] The results of Example 3 above confirmed that when the norovirus GII.17 antigen protein is expressed as a fusion protein with the existing RID, it maintains its activity as a fusion partner that helps the target protein fold. As a result, the soluble expression level is excellent, and protein misfolding is overcome. Therefore, the protein structure is stable, and the self-folding and assembly ability is improved. As a result, the VLP assembly efficiency and homogeneity of the finally purified norovirus GII.17 VP1 protein are excellent. Furthermore, the performance of antibody inhibition induced by the corresponding VLP inoculation and the binding of HBGA, which plays a core role in norovirus intracellular infection, is outstanding.

[0133] [Example 4]

[0134] Preparation of norovirus GII.3 VP1 virus-like particles (VLPs) with added N-terminal expression enhancers for high efficiency and soluble expression, and soluble enhancement partners.

[0135] <Example 4-1>

[0136] To prepare virus-like particles composed of norovirus GII.3 VP1 protein, the VP1 gene (Norovirus Hu / GII.3 / CHDC2005 / 1975 / US, NCBI access number: HM072045.1), a source of norovirus GII.3 protein, was utilized. The norovirus antigen VP1 consists of 547 amino acids, with a protein molecular weight of approximately 59.7 kDa. Based on this protein sequence, a gene sequence (SEQ ID NO. 10) was synthesized in *E. coli* after codon optimization. (The gene was synthesized by Bionics, Japan.)

[0137] Figure 4a In this study, a fusion protein was prepared by binding an EE domain (MSAVKAA (SEQ ID NO. 2)) and an SE domain (SEQ ID NO. 3) to the N-terminus of a protein, which have the effects of increasing expression and solubility, or a mutant SE domain peptide (SEQ ID NO. 7) that is more than 50% similar to SEQ ID NO. 3 and contains K23A, K30A, and K31A mutations, and the soluble expression status was verified.

[0138] Reference Figure 4a It was confirmed that the fusion protein (RID(EE domain+m3c)-NoV GII.3 VP1) containing the mutant RID peptide with K23A, K30A, and K31A mutations exhibited a higher soluble(s) expression state compared to the fusion protein (RID-NoV GII.3 VP1) containing the unmutated RID.

[0139] Furthermore, as the linker for the ENLYFQG / S sequence optimized using the TEV cleavage site, a peptide with the amino acid sequence (GaSb)n (a≥1, b≥1, n≥1) was used and placed between the TEV cleavage sequence (ENLYFQ) and the aforementioned target protein (norovirus antigen protein) sequence. A schematic diagram of the fusion protein expression vector prepared in this embodiment is shown below. Figure 1a .

[0140] Genes with the above sequences were prepared using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector, based on the pET-9a-EE domain(MSAVKAA)-SEdomain(wt)-Nov GII.17 VP1 and pET-9a-EE domain(MSAVKAA)-m3c-Nov GII.17 VP1 vectors, which were used as recombinant complex protein expression vectors prepared in Examples 1-1 above. T7 RNA polymerase was expressed via IPTG, thereby adding a RID sequence to the existing N-terminus of the pET vector, which operates on the Lac operon and T7 promoter located on the DE3 genome, to increase solubility and expression rate. Further insertion was performed using KpnI and BamHI cleavage enzymes in the C-direction of the ligation site sequence used to enhance 6xHIS and TEV cleavage efficiency. It was confirmed that a linker was inserted between the NdeI and KpnI sequences in the cleavage enzyme site within the MCS to improve the cleavage efficiency of the RID, cleavage enzyme site, and TEV enzyme in this study. Between the KpnI and BamHI cleavage enzymes, DNA sequences configured as proteins fused with norovirus VP1 antigen proteins were inserted. In each gene, the pET vector and the inserted gene sequence were ligated by T4 DNA ligase. The bacterial clones generated by their DNA ligation reaction were analyzed by DNA sequence analysis to show sequences with codon-optimized nucleotides inserted.

[0141] The sequences of the recombinant complex expression vectors prepared in Example 4-1 are as follows.

[0142] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GII.3 VP1

[0143] [(SEQ ID NO. 2)-(SEQ ID NO. 3)-GT-6xHIS-TEV-GS-(SEQ ID NO. 10)]:

[0144] MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEINNEAMALEPVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRNSPGEVLLNLELGPEINPYLAHLARMYNGYAGGFEVQVVLAGNAFTAGKVIFAAIPPNFPIDNLSAAQITMCPHVIVDVRQLEPINLPMPDVRNNFFHYNQGSDSRLRLIAMLYTPLRANNSGDDVFTVSCRVLTRPSPDFSFNFLVPPTVESKTKPFTLPILTISEMSNSRFPVPIDSLHTSPTENIVVQCQNGRVTLDGELMGTTQLLPSQICAFRGTLTRSTSRAGDQADTATPRLFNYYWHIQLDNLNGTPYDPAEDIPAPLGTPDFRGKVFGVASQRNPDSTTRAHEAKVDTTSGRFAPKLGSLEISTESSDFDSNQPTRFTPVGIGVDNEADFQQWSLPDYSGQFTHNMNLAPAVAPNFPGEQLLFFRSQLPSSGGRSNGILDCLVPQEWVQHFYQESAPAQTQVALVRYVNPDTGRVLFEAKLHKLGFMTIAKNGDSPITVPPNGYFRFESWVNPFYTLAPMGTGNGRRRIQ

[0145] Name the complex prepared above as "RID-NoV GII.3 VP1".

[0146] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GII.3 VP1

[0147] [(SEQ ID NO. 2)-(SEQ ID NO. 7)-GT-6xHIS-TEV-GS-(SEQ ID NO. 10)]

[0148] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0149] -GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEINNEAMALEPVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRNSPGEVLLNLELGPEINPYLAHLARMYNGYAGGFEVQVVLAGNAFTAGKVIFAAIPPNFPIDNLSAAQITMCPHVIVDVRQLEPINLPMPDVRNNFFHYNQGSDSRLRLIAMLYTPLRANNSGDDVFTVSCRVLTRPSPDFSFNFLVPPTVESKTKPFTLPILTISEMSNSRFPVPIDSLHTSPTENIVVQCQNGRVTLDGELMGTTQLLPSQICAFRGTLTRSTSRAGDQADTATPRLFNYYWHIQLDNLNGTPYDPAEDIPAPLGTPDFRGKVFGVASQRNPDSTTRAHEAKVDTTSGRFAPKLGSLEISTESSDFDSNQPTRFTPVGIGVDNEADFQQWSLPDYSGQFTHNMNLAPAVAPNFPGEQLLFFRSQLPSSGGRSNGILDCLVPQEWVQHFYQESAPAQTQVALVRYVNPDTGRVLFEAKLHKLGFMTIAKNGDSPITVPPNGYFRFESWVNPFYTLAPMGTGNGRRRIQ

[0150] Name the complex prepared above as "RID(EE domain+m3c)-NoV GII.3 VP1".

[0151] <Example 4-2>

[0152] The two expression vectors described in Example 4-1 were transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed *E. coli* were cultured in 3 ml of LB medium containing 50 μg / ml kanamycin at 37°C and 250 rpm for 5 to 7 hours. Then, 1 ml of the culture was transferred to 10 ml of the medium, diluted 10-fold, and cultured further. After approximately 2 to 4 hours, when the O.D600 reached 0.5–0.7, 0.4 mM isopropyl-β-D-thiogalactoside (IPTG, Biosang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17 to 19 hours (maximum 21 hours). The culture medium was centrifuged to obtain only the precipitated *E. coli*, which was then cryopreserved at -80°C. For each 3 ml volume of the harvested *E. coli*, 0.3 ml of a dissolution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added. After ultrasonic pulverization, the total lysate (T) was separated into precipitate (P) and soluble lysate (S) by centrifugation. SDS-PAGE analysis confirmed the separation of the precipitate and supernatant. Figure 4a As shown, the Norovirus GII.3 VP1 protein RID (EE domain+m3c)-NoV GII.3 VP1 fusion protein is formed by adding a mutant RID with an RNA binding site to the N-terminus. Figure 4a (Right side) shows highly efficient soluble expression.

[0153] <Example 4-3>

[0154] To obtain the RID (EE domain+m3c)-NoV GII.3 VP1 fusion protein, the same culture method as in Examples 1-2 above, which confirmed efficient soluble expression, was used. Cells obtained by inducing expression in 500 ml of *E. coli* culture were resuspended in 75 ml of lysis buffer A [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole]. The cell resuspension was then disrupted using an ultrasonic cell disruptor. The dissolved cells were centrifuged at 13500 rpm for 10 minutes at 4°C, and the supernatant was collected and subjected to Ni+ affinity chromatography using an AKTA (GE Healthcare) analyzer. First, a Ni Sepharose High Performance (Cytiva) column was equilibrated using buffer A (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole). Cell lysate supernatant was then loaded into the equilibration column at a flow rate of 3–5 ml / min. Norovirus GII.3 VP1 protein was eluted and obtained using buffer A and buffer B containing 300 mM imidazole [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole], with imidazole injected in a linear concentration gradient from 10 mM to 300 mM.

[0155] The separation status of the target protein was confirmed by SDS-PAGE of the above eluents. The corresponding fractionated eluents were dialyzed overnight (12 to 16 hours) at 4°C in dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol]. The N-terminal RID and norovirus antigen protein VP1 were cleaved using TEV cleavage enzyme.

[0156] To separate and purify the removed target proteins, such as RID, which are N-terminal fusion partners, a Ni Sepharose High Performance (Cytiva) column was used. The column was equilibrated with the same solution A as the dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% 2-mercaptoethanol] and loaded into the column at a flow rate of 3–5 ml / min. The removed NoV GII.-3 target proteins, such as RID, which are N-terminal fusion partners, were recovered in a form that was not adsorbed onto the column and eluted.

[0157] To increase the final purity of the target protein, ion exchange chromatography was performed using AKTA (GE Healthcare). The column (Fractogel EMD DEAE(M), Merck Millipore) was equilibrated with buffer solution A [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol] and loaded at a flow rate of 1 ml / min. The target protein was eluted by passing it through buffer solutions AA and BB [50 mM Tris-HCl (pH 8.5), 1 M NaCl, 5% glycerol, 0.1% β-mercaptoethanol] and by injecting NaCl with a linear concentration gradient from 10 mM to 1 M. The elution was achieved by fractional distillation of 1 ml each. The resulting purification status and purity are as follows: Figure 4b , Figure 4c , Figure 4d The concentration of the finally purified protein was quantified using BSA (Soren Amresco, Ohio, USA). (See reference...) Figures 4b to 4d It is evident that separating the RID protein bound within the fusion protein allows for effective purification of the target protein.

[0158] <Example 4-4>

[0159] The purified norovirus VP1 protein was prepared in a state containing 10% glycerol and was in the final state of assembly buffer [100 mM potassium phosphate, pH 5.8, 150 mM NaCl, 10% glycerol]. The buffer was exchanged overnight (12 to 16 hours) and stored at 4°C. The concentration of the finally purified protein was quantified using BSA (Amresco, Soren, Ohio, USA).

[0160] <Example 4-5>

[0161] To confirm the overall diameter distribution of virus-like particles (VLPs) self-assembled from the purified target protein after cleavage of the RID in the fusion protein prepared in Examples 4-4 (a norovirus VP1 fusion protein containing the RID mutant protein (RID(EE domain+m3c)-NoVGII.3 VP1 fusion protein)), dynamic light scattering (DLS) was used for analysis. In the analysis of the above complexes, 1 mL of sample was added to a cuvette at 16°C using DLS (Particular Systems, Zetasizer Nano Family) and the measurement was performed. In the dynamic light scattering analysis of cells, the total intensity and mass of each region were used to confirm the distribution in the image. Figure 4e It was confirmed that the particles formed in the H hydrodynamic radius with DLS have a diameter distribution of 30-40 nm.

[0162] <Examples 4-6>

[0163] The morphology of virus-like particles (VLPs) assembled from the target protein purified from the fusion protein (RID(EE domain+m3c)-NoV GII.3 VP1 fusion protein) according to Examples 4-4 was observed using electron microscopy. The purified norovirus VLPs were first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, dried at room temperature for 10 minutes, and then photographed using a transmission electron microscope (120kV, TEM; Talos L120C, FEI, Czech Republic). Figure 4fAs shown, the purified and assembled norovirus VP1 protein was confirmed to form a virus-like particle (VLP). The diameter of the confirmed VLP was confirmed to be between 30 and 40 nm, the same as that of the norovirus particle, forming a highly homogeneous spherical VLP.

[0164] The results of Example 4 also confirm that, similar to those of Example 2, when the mutant RID and norovirus antigen protein of the present invention are fused, more efficient target protein expression, appropriate folding induction, and improved VLP assembly ability are achieved in Escherichia coli.

[0165] [Example 5]

[0166] The immunogenicity of the formed GII.3 VLP was confirmed using mRID.

[0167] <Example 5-1>

[0168] To determine the induction capacity of norovirus GII.3 antigen-specific antibodies, virus-like particles (VLPs) prepared according to the present invention (VLPs composed of antigen proteins purified after cleavage of the RID in the fusion protein (RID(EE domain +m3c)-NoV GII.3 VP1) of the present invention) were injected intramuscularly into a group of 7-week-old Sprague Dawley (SD) rats (5 rats / group) at a concentration of 500 μL. Figure 5a Using a syringe fitted with a 26-gauge needle, the prescribed dosage (500 μL / head) was injected into both thighs of the animal, mixed with norovirus GII.3 VLP antigen and 0.5 mg of the immunostimulant aluminum hydroxide. The vaccine was administered three times every two weeks for immunization. Approximately 1 mL of whole blood was collected via the jugular vein at predetermined times and injected into a vacuum blood collection tube containing a clotactivator.

[0169] To determine the target protein-specific antibody titers in serum obtained from each animal experimental group, an enzyme-linked immunosorbent assay (ELISA) was performed. Norovirus GII.3 VLP was added to 96-well plates (High Binding Capacity (HBC) NeutrAvidin plates, Thermo Scientific™) at a concentration of 0.5 μg / mL, 100 μL per well. After coating overnight at 4°C, 200 μL of 3% BSA solution was added, and the plates were incubated at 37°C for 2 hours and then blocked. The plates were washed with PBST, and the immune serum was diluted (1:1600 dilution), 100 μL per well, and incubated at 37°C for 1 hour and 30 minutes. After washing the plates with PBST, to detect antibodies in the serum bound to GII.3 VLP, antibodies against goat-derived rat IgG (Southern Biotech 1030-05, Goat-anti-mouse IgG HRP) were administered at 37°C for 1 hour. Subsequently, TMB (sigma) was used to detect antibodies. After the T0440 solution was incubated at room temperature for 5 minutes, the colorimetric reaction was stopped with 0.5M sulfuric acid solution, and the absorbance was measured at 450 nm. The GII.3 VLP-specific antibody titer in post-vaccination blood samples was confirmed by measuring 8000-fold dilutions from the experimental groups (0.2 μg / head, 1 μg / head, 5 μg / head, 10 μg / head, and 20 μg / head). Figure 5ba As shown, after one vaccination, specific IgG antibody titers were confirmed in the groups administering doses of 1 μg or higher. Starting after two vaccinations, high-specific IgG antibody titers were confirmed in all groups administering doses. Figure 5bb , Figure 5bc ).

[0170] <Example 5-2>

[0171] To determine the immunosuppressive effect of GII.3 VLP antigen (composed of the antigen protein purified after RID cleavage from the RID(EE domain+m3c)-NoVGII.3VP1 fusion protein of this invention), Figure 5c The degree of binding between Nov GII.3 VLP and HBGA was determined, and an HBGA blocking assay was performed on the obtained immune serum according to the schedule after vaccination. Figure 5cIn 96-well plates coated with streptavidin (High Binding Capacity (HBC) NeutrAvidin plates (Thermo Scientific™), USA), Lea-PAA-biotin (01-035) (Glycotech), which has high binding affinity for GII.3, was added at a concentration of 10 μg / ml and incubated at room temperature for 5 hours. First, second, and third immune sera, used to confirm the inhibitory efficacy of VLP antigen binding to HBGA, were sequentially diluted 100-fold to 2-fold in PBS and reacted with appropriately diluted GII.3 VLP protein at 37°C for 1 hour and 30 minutes. Serum and GII.3 VLP reaction solution were added at 100 μl per well to the plate reacted with HBGA and incubated at 4°C for 18 hours. The plates were washed with PBST, and rabbit NoVGII.3 polyclonal IgG was added using PBS. Dilute Ab 2000-fold, add 100 μL to each well, and react at room temperature for 1 hour and 30 minutes. After washing with PBST, dilute goat-derived rabbit IgG (Goat-anti rabbit IgG HRP) 30000-fold in PBS, add 100 μL to each well, and treat at room temperature for 1 hour and 30 minutes. Then, develop the color with TMB solution at room temperature for 5 minutes, stop the color development reaction with 0.5 M sulfuric acid solution, and measure the absorbance at 450 nm.

[0172] Figure 5c To assess the efficacy of HBGA binding inhibition in immune serum (using Lea synthetic carbohydrates), HBGA was administered at concentrations of 5 μg / head (low concentration), 10 μg / head (medium concentration), and 20 μg / head (high concentration) 14 days after single, double, and triple vaccinations. GII.3 analysis showed that after single vaccination, HBGA binding inhibition efficacy of ≥1:200 was confirmed at all doses. After double vaccination, significantly increased HBGA binding inhibition capacity of ≥1:1500 was confirmed at all doses compared to single administration. After triple vaccination, high HBGA binding inhibition capacity of ≥1:2500 was confirmed at all doses. After single vaccination, similar HBGA binding inhibition effects were observed in all dose groups. After double vaccination, higher efficacy was induced compared to single vaccination. Higher HBGA binding inhibition efficacy was induced in the 10 μg and 20 μg administration groups compared to the 5 μg administration group. Three doses induced higher HBGA binding inhibition efficacy, showing overall saturation regardless of dose, thus confirming the high immunogenicity of the antigen.

[0173] The results of Example 5 also confirmed that, similar to those of Example 3, when the norovirus GII.3 antigen protein of the present invention is expressed as a fusion protein with existing RID, it maintains activity as a fusion partner that helps the target protein fold, thereby achieving excellent soluble expression levels, overcoming protein misfolding, and thus ensuring protein structural stability and improving self-folding and assembly capabilities. Consequently, the VLP assembly efficiency and homogeneity of the finally purified norovirus GII.3 VP1 protein are excellent, and the antibody inhibition induced by the corresponding VLP inoculation exhibits outstanding binding performance to HBGA, which plays a core role in intracellular infection of norovirus.

[0174] [Example 6]

[0175] Prepare VP1 virus-like particles (VLPs) based on the genotype of norovirus antigen target proteins purified from fusion proteins.

[0176] To prepare virus-like particles composed of the target protein (norovirus antigen protein) purified from a norovirus VP1 protein fusion protein with a RID sequence attached to the N-terminus for efficient soluble expression, genes from various norovirus genotypes GI.1 (Norovirus Hu / GI.1 / 8McIII / 1973 / USA, NCBI access number: NC_001959.2), GI.3 (Norwalk-like virus NLV / Honolulu / 219 / 1992 / US, NCBI access number: AF414403), and GII.2 (Norovirus GII.2 strain Env / CHN / 2016 / GII.P16-GII.2 / BJSMQ, NCBI access number: NC_039476) that are derived from VP1 proteins were utilized.

[0177] <Example 6-1>

[0178] Figure 6a In this study, the VP1 gene, derived from the protein of norovirus genotype GI.1 (Norovirus Hu / GI.1 / 8McIII / 1973 / USA, NCBI access number: NC_001959.2), was utilized. The norovirus antigen VP1 consists of approximately 500 amino acids, with a protein molecular weight of approximately 55-60 kDa. Based on this protein sequence, a gene sequence (SEQ ID NO. 12) was synthesized in *E. coli* after codon optimization. (The gene was synthesized by Bionics, Japan.)

[0179] Genes with the above sequences were prepared using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector, based on the pET-9a-EE domain(MSAVKAA)-SEdomain(WT)-Nov GII.17 VP1 and pET-9a-EE domain(MSAVKAA)-m3c-Nov GII.17 VP1 vectors, which were used as recombinant complex protein expression vectors prepared in Examples 1-1 above. T7 RNA polymerase was expressed via IPTG, thereby adding a RID sequence to the existing N-terminus of the pET vector, which operates on the Lac operon and T7 promoter located on the DE3 genome, to increase solubility and expression rate. The gene was then digested and subcloned using KpnI and BamHI cutting enzymes in the C-direction of the linker sequence used to enhance 6xHIS and TEV cleavage efficiency, and then reinserted. It was confirmed that a linker was inserted between the NdeI and KpnI sequences in the cleavage enzyme site within the MCS to improve the cleavage efficiency of the RID, cleavage enzyme site, and TEV enzyme in this study. Between the KpnI and BamHI cleavage enzymes, DNA sequences configured as proteins fused with norovirus VP1 antigen proteins were inserted. In each gene, the pET vector and the inserted gene sequence were ligated by T4 DNA ligase. The bacterial clones generated by their DNA ligation reaction were analyzed by DNA sequence analysis to insert codon-optimized nucleotide sequences.

[0180] Recombinant proteins were prepared by fusing RID EE domain peptides (MSAVKAA (SEQ ID NO. 2)) and RID SE domain peptides (SEQ ID NO. 3) with the N-terminus of the protein, which have the effects of increasing expression and solubility, or mutant RID peptides (SEQ ID NO. 7) that are more than 50% similar to SEQ ID NO. 3 and contain K23A, K30A, and K31A mutations, and the soluble expression status was verified.

[0181] Furthermore, as a linker for the ENLYFQG / S optimized sequence of the TEV cleavage site, a peptide with the amino acid sequence (GaSb)n (a≥1, b≥1, n≥1) was used and placed between the TEV cleavage sequence and the target protein (norovirus antigen protein) sequence.

[0182] The recombinant complex expression vectors confirmed in Example 4-1 using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector containing the target protein with the above sequence have the following two sequences.

[0183] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GI.1 VP1

[0184] [(SEQ ID NO. 2)-(SEQ ID NO. 3)-GT-6xHIS-TEV-GS-(SEQ ID NO. 12)]

[0185] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-MMMASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWII NNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFN FLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGV LSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR

[0186] The composite prepared above was named "RID-NoV GI.1 VP1".

[0187] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GI.1 VP1

[0188] [(SEQ ID NO. 2)-(SEQ ID NO. 7)-GT-6xHIS-TEV-GS-(SEQ ID NO. 12)]

[0189] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0190] -GT-HHHHHH-ENLYFQ-GSMMMASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGVLSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR

[0191] Name the complex prepared above as "RID(EE domain+m3c)-NoV GI.1 VP1".

[0192] <Example 6-2>

[0193] The two expression vectors described in Example 5-1 were transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed *E. coli* were cultured in 3 ml of LB medium containing 50 μg / ml kanamycin at 37°C and 250 rpm for 5 to 7 hours. Then, 1 ml was transferred to 10 ml of the medium, diluted 10-fold, and cultured further. After approximately 2 to 4 hours, when the O.D600 reached 0.5–0.7, 0.4 mM isopropyl-β-D-thiogalactoside (IPTG, Biosang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17 to 19 hours (maximum 21 hours). The culture medium was centrifuged to obtain only the precipitated *E. coli*, which was then cryopreserved at -80°C. For each 3 ml volume of the harvested *E. coli*, 0.3 ml of a dissolution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added. After ultrasonic pulverization, the total lysate (T) was separated into precipitate (P) and soluble lysate (S) by centrifugation. SDS-PAGE analysis confirmed the separation of the precipitate and supernatant. Figure 5a As shown, norovirus GI.1 VP1 protein with mutant RID added to the N-terminus exhibits highly efficient soluble expression.

[0194] <Example 6-3>

[0195] Figure 6b The gene for VP1, derived from the protein of norovirus genotype GI.3 (Norwalk-like virus NLV / Honolulu / 219 / 1992 / US, NCBI access number: AF414403), was utilized. The norovirus antigen VP1 consists of approximately 500 amino acids, with a protein molecular weight of approximately 55-60 kDa. Based on this protein sequence, a gene sequence (SEQ ID NO. 13) was synthesized in *E. coli* after codon optimization. (The gene was synthesized by Bionics, Japan.)

[0196] Genes with the above sequences were prepared using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector, and based on the pET-9a-MSAVKAA-RID-Nov GII.17 VP1 and pET-9a-MSAVKAA-Nov GII.17 vectors, which were used as the recombinant complex protein expression vectors prepared in Example 1 above. T7 RNA polymerase was expressed via IPTG, thereby adding a RID sequence to the existing N-terminus of the pET vector, which operates on the Lac operon and T7 promoter located on the DE3 genome, to increase solubility and expression rate. The sequences were then cut, subcloned, and re-inserted using KpnI and BamHI cutting enzymes in the C-direction of the ligation site sequence used to enhance 6xHIS and TEV cleavage efficiency. It was confirmed that a linker was inserted between the NdeI and KpnI sequences in the cleavage enzyme site within the MCS to improve the cleavage efficiency of the RID, cleavage enzyme site, and TEV enzyme in this study. Between the KpnI and BamHI cleavage enzymes, DNA sequences configured as proteins fused with norovirus VP1 antigen proteins were inserted. In each gene, the pET vector and the inserted gene sequence were ligated by T4 DNA ligase. The bacterial clones generated by their DNA ligation reaction were analyzed by DNA sequence analysis and found to have codon-optimized nucleotide sequences inserted.

[0197] Recombinant proteins were prepared by fusing MSAVKAA (SEQ ID NO. 2) and RID peptide (SEQ ID NO. 3) with the N-terminus of the protein, which have the effects of increasing expression and solubility, or a mutant SE domain peptide (SEQ ID NO. 7) that is more than 50% similar to SEQ ID NO. 3 and contains K23A, K30A, and K31A mutations, and the soluble expression status was verified.

[0198] Furthermore, as a linker for the ENLYFQG / S optimized sequence of the TEV cleavage site, a peptide with the amino acid sequence (GaSb)n (a≥1, b≥1, n≥1) was used and placed between the TEV cleavage sequence and the target protein sequence (norovirus antigen protein).

[0199] The recombinant complex expression vectors confirmed in Examples 5-3 using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 3) expression vector containing the target protein with the above sequence are as follows:

[0200] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GI.3 VP1

[0201] [(SEQ ID NO. 2)-(SEQ ID NO. 3)-GT-6xHIS-TEV-GS-(SEQ ID NO. 13)]

[0202] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-MMMASKDAPPNMDGTSGAGQLVPEANTAEPISMEPVAGAATAAATAGQVNMIDPWIMNNYVQAPQGEFTISPNNTPGDILFDLQLGPHLNPFLSHLAQMYNGWVGNMKVKVLLAGNAFTAGKIIISCIPPGFAAQNISIAQATMFPHVIADVRVLEPIEVPLEDVRNVLFHNNDNTPTMRLVCMLYTPLRASGSSSGTDPFVIAGRVLTCPSPDFSLLFLVPPNVEQKTKPFSVPNLPLNTLSNSRVPSLIKSMMVSRDHGQMVQFQNGRVALDGQLQGTTPTSASQLCKIRGSVFHANGGNGYNPTELDGSPYHAFESPAPIGFPDLGECDWHMEASPTTQFDTGDVIKQINVKQESAFAPHLGTIQADGLSDVSVNTNMIAKLGWVSPVSDGHRGNVDPWVIPRYGSTLTEAAQLAPPIYPPGFGEAIVFFMSDFPIAHGTNGLSVPCTIPQEFVTHFVNEQAPTRGEAALLHYLDPDTHRNLGEFKLYPEGFMTCVPNSSGTGPQTLPINGVFVFVSWVSRFYQLKPVGTAGPARRLGIRRS

[0203] Name the complex prepared above as "RID-NoV GI.3 VP1".<s

[0204] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GI.3 VP1

[0205] [(SEQ ID NO. 2)-(SEQ ID NO. 7)-GT-6xHIS-TEV-GS-(SEQ ID NO. 13)]

[0206] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0207] -GT-HHHHHH-ENLYFQ-GS-MMMASKDAPPNMDGTSGAGQLVPEANTAEPISMEPVAGAATAAATAGQVNMIDPWIMNNYVQAPQGEFTISPNNTPGDILFDLQLGPHLNPFLSHLAQMYNGWVGNMKVKVLLAGNAFTAGKIIISCIPPGFAAQNISIAQATMFPHVIADVRVLEPIEVPLEDVRNVLFHNNDNTPTMRLVCMLYTPLRASGSSSGTDPFVIAGRVLTCPSPDFSLLFLVPPNVEQKTKPFSVPNLPLNTLSNSRVPSLIKSMMVSRDHGQMVQFQNGRVALDGQLQGTTPTSASQLCKIRGSVFHANGGNGYNPTELDGSPYHAFESPAPIGFPDLGECDWHMEASPTTQFDTGDVIKQINVKQESAFAPHLGTIQADGLSDVSVNTNMIAKLGWVSPVSDGHRGNVDPWVIPRYGSTLTEAAQLAPPIYPPGFGEAIVFFMSDFPIAHGTNGLSVPCTIPQEFVTHFVNEQAPTRGEAALLHYLDPDTHRNLGEFKLYPEGFMTCVPNSSGTGPQTLPINGVFVFVSWVSRFYQLKPVGTAGPARRLGIRRS

[0208] Name the complex prepared above as "RID(EE domain+m3c)-NoV GI.3 VP1".

[0209] <Example 6-4>

[0210] The two expression vectors described in Examples 6-3 above were transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed *E. coli* were cultured in 3 ml of LB medium containing 50 μg / ml kanamycin at 37°C and 250 rpm for 5 to 7 hours. Then, 1 ml was transferred to 10 ml of the medium, diluted 10-fold, and cultured further. After approximately 2 to 4 hours, when the O.D600 reached 0.5–0.7, 0.4 mM isopropyl-β-D-thiogalactoside (IPTG, Biosang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17 to 19 hours (maximum 21 hours). The culture medium was centrifuged to obtain only the precipitated *E. coli*, which was then cryopreserved at -80°C. For each 3 ml volume of the harvested *E. coli*, 0.3 ml of a solubilization buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added. After ultrasonic pulverization, the total lysate (T) was separated into precipitate (P) and supernatant (S) by centrifugation. SDS-PAGE analysis, as shown in Figure 5b, confirmed that norovirus GI.3 VP1 protein with the mutant RID added to the N-terminus exhibited highly soluble expression.

[0211] <Example 6-5>

[0212] Figure 6c The study utilized the VP1 gene derived from norovirus genotype GII.2 (Norovirus GII.2 strain Env / CHN / 2016 / GII.P16-GII.2 / BJSMQ, NCBI access number: NC_039476). The norovirus antigen VP1 consists of approximately 500 amino acids, with a protein molecular weight of approximately 55-60 kDa. Based on this protein sequence, a gene sequence (SEQ ID NO. 11) was synthesized from *E. coli* after codon optimization. (The gene was synthesized by Bionics, Japan.)

[0213] Genes with the above sequences were prepared using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector, based on the pET-9a-MSAVKAA-RID-Nov GII.17 VP1 and pET-9a-MSAVKAA-m3c-Nov GII.17 vectors, which were used as recombinant complex protein expression vectors prepared in Example 1 above. T7 RNA polymerase was expressed via IPTG, thereby adding a RID sequence to the existing N-terminus of the pET vector, which operates on the Lac operon and T7 promoter located on the DE3 genome, to increase solubility and expression rate. The sequences were then cut, subcloned, and re-inserted using KpnI and BamHI cutting enzymes in the C-direction of the ligation site sequence used to enhance 6xHIS and TEV cutting efficiency. It was confirmed that a linker was inserted between the NdeI and KpnI sequences in the cleavage enzyme site within the MCS to improve the cleavage efficiency of the RID, cleavage enzyme site, and TEV enzyme in this study. Between the KpnI and BamHI cleavage enzymes, DNA sequences configured as proteins fused with norovirus VP1 antigen proteins were inserted. In each gene, the pET vector and the inserted gene sequence were ligated by T4 DNA ligase. The bacterial clones generated by their DNA ligation reaction were analyzed by DNA sequence analysis and found to have codon-optimized nucleotide sequences inserted.

[0214] A recombinant protein was prepared by fusing an EE domain peptide (MSAVKAA (SEQ ID NO. 2)) and an SE domain peptide (SEQ ID NO. 3) with increased expression and solubility at the N-terminus of a target protein (norovirus antigen protein), or a mutant SE domain peptide (SEQ ID NO. 7) that is more than 50% similar to SEQ ID NO. 3 and contains K23A, K30A, and K31A mutations, thereby verifying the soluble expression status.

[0215] Furthermore, as a linker for the ENLYFQG / S optimized sequence of the TEV cleavage site, a peptide with the amino acid sequence (GaSb)n (a≥1, b≥1, n≥1) was used and placed between the TEV cleavage sequence and the target protein sequence (norovirus antigen protein).

[0216] The recombinant complex expression vectors confirmed in Examples 6-5 using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO. 1) expression vector containing the target protein with the above sequence are as follows:

[0217] MSAVKAA-RID-GT-6xHIS-TEV-GS-NoV GII.2 VP1

[0218] [(SEQ ID NO. 2)-(SEQ ID NO. 3)-GT-6xHIS-TEV-GS-(SEQ ID NO. 11)]

[0219] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-MKMASNDAAPSTDGAAGLVPESNNEVMALEPVAGAALAAPVTGQTNIIDPWIRANFVQAPNGEFTVSPRNAPGEVLLNLELGPELNPYLAHLARMYNGYAGGMEVQVMLAGNAFTAGKLVFAAVPPHFPVENLSPQQITMFPHVIIDVRTLEPVLLPLPDVRNNFFHYNQKDDPKMRIVAMLYTPLRSNGSGDDVFTVSCRVLTRPSPDFDFTYLVPPTVESKTKPFTLPILTLGELSNSRFPVSIDQMYTSPNEIISVQCQNGRCTLDGELQGTTQLQVSGICAFKGEVTAHLHDNDHLYNVTITNLNGSPFDPSEDIPAPLGVPDFQGRVFGIISQRDKHNSPGHNEPANRGHDAVVPTYTAQYTPKLGQIQIGTWQTDDLTVNQPVKFTPVGLNDTEHFNQWVVPRYAGALNLNTNLAPSVAPVFPGERLLFFRSYIPLKGGYGNPAIDCLLPQEWVQHFYQEAAPSMSEVALVRYINPDTGRALFEAKLHRAGFMTVSSNTSAPVVVPANGYFRFDSWVNQFYSLAPMGTGNGRRRVQ

[0220] Name the complex prepared above as "RID-NoV GII.2 VP1".

[0221] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GII.2 VP1

[0222] [(SEQ ID NO. 2)-(SEQ ID NO. 7)-GT-6xHIS-TEV-GS-(SEQ ID NO. 11)]

[0223] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0224] -GT-HHHHHH-ENLYFQ-GS-MKMASNDAAPSTDGAAGLVPESNNEVMALEPVAGAALAAPVTGQTNIIDPWIRANFVQAPNGEFTVSPRNAPGEVLLNLELGPELNPYLAHLARMYNGYAGGMEVQVMLAGNAFTAGKLVFAAVPPHFPVENLSPQQITMFPHVIIDVRTLEPVLLPLPDVRNNFFHYNQKDDPKMRIVAMLYTPLRSNGSGDDVFTVSCRVLTRPSPDFDFTYLVPPTVESKTKPFTLPILTLGELSNSRFPVSIDQMYTSPNEIISVQCQNGRCTLDGELQGTTQLQVSGICAFKGEVTAHLHDNDHLYNVTITNLNGSPFDPSEDIPAPLGVPDFQGRVFGIISQRDKHNSPGHNEPANRGHDAVVPTYTAQYTPKLGQIQIGTWQTDDLTVNQPVKFTPVGLNDTEHFNQWVVPRYAGALNLNTNLAPSVAPVFPGERLLFFRSYIPLKGGYGNPAIDCLLPQEWVQHFYQEAAPSMSEVALVRYINPDTGRALFEAKLHRAGFMTVSSNTSAPVVVPANGYFRFDSWVNQFYSLAPMGTGNGRRRVQ

[0225] Name the complex prepared above as "RID(EE domain+m3c)-NoV GII.2 VP1".

[0226] <Example 6-6>

[0227] The two expression vectors described in Examples 6-5 were transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed *E. coli* were cultured in 3 ml of LB medium containing 50 μg / ml kanamycin at 37°C and 250 rpm for 5 to 7 hours. Then, 1 ml was transferred to 10 ml of the medium, diluted 10-fold, and cultured further. After approximately 2 to 4 hours, when the O.D600 reached 0.5–0.7, 0.4 mM isopropyl-β-D-thiogalactoside (IPTG, Biosang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17 to 19 hours (maximum 21 hours). The culture medium was centrifuged to obtain only the precipitated *E. coli*, which was then cryopreserved at -80°C. For each 3 ml volume of the harvested *E. coli*, 0.3 ml of a dissolution buffer (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added. After ultrasonic pulverization, the total lysate (T) was separated into precipitate (P) and soluble lysate (S) by centrifugation. SDS-PAGE analysis confirmed the separation of the precipitate and the precipitate. Figure 5c As shown, the norovirus GII.2 VP1 protein with the mutant RID added to the N-terminus (fusion protein) exhibits highly efficient soluble expression.

[0228] <Example 6-7>

[0229] For the norovirus GI.1, GI.3, and GII.2 VP1 obtained in Example 6 above, the purified norovirus VP1 protein, after undergoing the same purification process as in the above examples, was stored overnight (12 to 16 hours) in assembly buffer containing 10% glycerol at 4°C. To confirm the overall diameter distribution of the assembled virus-like particles (VLPs), dynamic light scattering (DLS) analysis was performed. In the analysis of the complexes, 1 mL of sample was added to a cuvette at 16°C using DLS (Particular Systems, Zetasizer NanoFamily) and measured. In the dynamic light scattering analysis of cells, the total intensity and mass of each region were used to confirm the image. Furthermore, the morphology of the virus-like particles (VLPs) was observed using electron microscopy. The purified norovirus VLP was first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, dried at room temperature for 10 minutes, and then photographed using a transmission electron microscope (TEM; Talos L120C, FEI, Czech Republic). Figures 6da to 6dc As shown, the purified and assembled norovirus VP1 protein exhibits a particle size distribution of 30–40 nm in diameter within the hydrodynamic radius of the DLS, and forms VLPs. It was confirmed that the VLPs observed in TEM have diameters between 30 and 40 nm, consistent with norovirus particle diameters, and form highly homogeneous spherical VLPs.

[0230] It has been confirmed that when the mutant RID of the present invention is fused with the norovirus antigen protein, it is intuitively important for expressing the target protein more efficiently in E. coli, inducing proper folding, and improving VLP assembly capabilities.

[0231] The embodiments of the present invention have been described above, but it will be understood by those skilled in the art to which this invention pertains that the present invention can be implemented in different specific forms without changing its technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not limiting.

[0232] Sequence List

[0233] SEQ ID NO. 1

[0234] Vector diagram (pET-9a)

[0235] ttctcatgtttgacagcttatcatcgataagctttaatgcggtagtttatcacagttaaattgctaacgcagtcaggcaccgtgtatgaaatctaacaatgcgctcatcgtcatcctcggcaccgtcaccctggatgctgtaggcataggcttggttatgccggtactgccgggcctcttgcgggatatcgtccattccgacagcatcgccagtcactatggcgtgctgctagcgctatatgcgttgatgcaatttctatgcgcacccgttctcggagcactgtccgaccgctttggccgccgcccagtcctgctcgcttcgctacttggagccactatcgactacgcgatcatggcgaccacacccgtcctgtggata

[0236] tccggatatagttcctcctttcagcaaaaaacccctcaagacccgtttagaggccccaaggggttatgctagttattgctcagcggtggcagcagccaactcagcttcctttcgggctttgttagcagccggatccgcgacccatttgctgtccaccagtcatgctagccatatgtatatctccttcttaaagttaaacaaaattatttctagagggaaaccgttgtggtctccctatagtgagtcgtattaatttcgcgggatcgagatctcgatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcggg

[0237] ctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaccgatgcccttgagagccttcaacccagtcagctccttccggtgggcgcggggcatgactatcgtcgccgcacttatgactgtcttctttatcatgcaactcgtaggacaggtgccggcagcgctctgggtcattttcggcgaggaccgctttcgctggagcgcgacgatgatcggcctgtcgcttgcggtattcggaatcttgcacgccc

[0238] tcgctcaagccttcgtcactggtcccgccaccaaacgtttcggcgagaagcaggccattatcgccggcatggcggccgacgcgctgggctacgtcttgctggcgttcgcgacgcgaggctggatggccttccccattatgattcttctcgcttccggcggcatcgggatgcccgcgttgcaggccatgctgtccaggcaggtagatgacgaccatcagggacagcttcaaggatcgctcgcggctcttaccagcctaacttcgatcactggaccgctgatcgtcacggcgatttatgccgcctcggcgagcacatggaacgggttggcatggattgtaggcgccgccctataccttgtctgcctccccgcgttgcgtcgcggtgc

[0239] atggagccgggccacctcgacctgaatggaagccggcggcacctcgctaacggattcaccactccaagaattggagccaatcaattcttgcggagaactgtgaatgcgcaaaccaacccttggcagaacatatccatcgcgtccgccatctccagcagccgcacgcggcgcatctcgggcagcgttgggtcctggccacgggtgcgcatgatcgtgctcctgtcgttgaggacccggctaggctggcggggttgccttactggttagcagaatgaatcaccgatacgcgagcgaacgtgaagcgactgctgctgcaaaacgtctgcgacctgagcaacaacatgaatggtcttcggtttccgtgtttcgtaaagtctgg

[0240] aaacgcggaagtcagcgccctgcaccattatgttccggatctgcatcgcaggatgctgctggctaccctgtggaacacctacatctgtattaacgaagcgctggcattgaccctgagtgatttttctctggtcccgccgcatccataccgccagttgtttaccctcacaacgttccagtaaccgggcatgttcatcatcagtaacccgtatcgtgagcatcctctctcgtttcatcggtatcattacccccatgaacagaaatcccccttacacggaggcatcagtgaccaaacaggaaaaaaccgcccttaacatggcccgctttatcagaagccagacattaacgcttctggagaaactcaacgagctggacgcggatgaaca

[0241] ggcagacatctgtgaatcgcttcacgaccacgctgatgagctttaccgcagctgcctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggcgcagccatgacccagtcacgtagcgatagcggagtgtatactggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgt

[0242] tcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaag

[0243] ctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacg

[0244] ctcagtggaacgaaaactcacgttaagggattttggtcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcttgctcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgattgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgcctcttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccactg

[0245] cgatccccgggaaaacagcattccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcattcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgtctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataagcttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccgata

[0246] ccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaagaa

[0247] SEQ ID NO. 2

[0248] RID EE domain

[0249] MSAVKAA

[0250] SEQ ID NO. 3

[0251] RID SE domain

[0252] AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0253] SEQ ID NO. 4

[0254] RID SE domain m9

[0255] AAVQAAEVKVDGSEPKLSANELAARLAAEAAVAEAEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV

[0256] SEQ ID NO. 5

[0257] RID SE domain m3a

[0258] AAVQAAEVKVDGSEPKLSANELKRRLKAEKKVAEKEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV

[0259] SEQ ID NO. 6

[0260] RID SE domain m3b

[0261] AAVQAAEVKVDGSEPKLSANELAARLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0262] SEQ ID NO. 7

[0263] RID SE domain m3c

[0264] AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0265] SEQ ID NO. 8

[0266] RID SE domain m4

[0267] AAVQAAEVKVDGSEPKLSKNELAARLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[0268] SEQ ID NO. 9

[0269] Norovirus genotype GII.17 (Norovirus Hu / GII.17 / HKG / 2014 / CUHK-NS-405; GeneID: KP902566.1)

[0270] MKMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0271] SEQ ID NO. 10

[0272] Norovirus genotype GII.3 (Norovirus Hu / GII.3 / CHDC2005 / 1975 / US; Gene ID: HM072045.1)

[0273] MKMASNDAAPSNDGAAGLVPEINNEAMALEPVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRNSPGEVLLNLELGPEINPYLAHLARMYNGYAGGFEVQVVLAGNAFTAGKVIFAAIPPNFPIDNLSAAQITMCPHVIVDVRQLEPINLPMPDVRNNFFHYNQGSDSRLRLIAMLYTPLRANNSGDDVFTVSCRVLTRPSPDFSFNFLVPPTVESKTKPFTLPILTISEMSNSRFPVPIDSLHTSPTENIVVQCQNGRVTLDGELMGTTQLLPSQICAFRGTLTRSTSRAGDQADTATPRLFNYYWHIQLDNLNGTPYDPAEDIPAPLGTPDFRGKVFGVASQRNPDSTTRAHEAKVDTTSGRFAPKLGSLEISTESSDFDSNQPTRFTPVGIGVDNEADFQQWSLPDYSGQFTHNMNLAPAVAPNFPGEQLLFFRSQLPSSGGRSNGILDCLVPQEWVQHFYQESAPAQTQVALVRYVNPDTGRVLFEAKLHKLGFMTIAKNGDSPITVPPNGYFRFESWVNPFYTLAPMGTGNGRRRIQ

[0274] SEQ ID NO. 11

[0275] Norovirus Genotype GII.2 (Norovirus GII.2 strain Env / CHN / 2016 / GII.P16-GII.2 / BJSMQ; Gene ID: NC_039476)

[0276] MKMASNDAAPSTDGAAGLVPESNNEVMALEPVAGAALAAPVTGQTNIIDPWIRANFVQAPNGEFTVSPRNAPGEVLLNLELGPELNPYLAHLARMYNGYAGGMEVQVMLAGNAFTAGKLVFAAVPPHFPVENLSPQQITMFPHVIIDVRTLEPVLLPLPDVRNNFFHYNQKDDPKMRIVAMLYTPLRSNGSGDDVFTVSCRVLTRPSPDFDFTYLVPPTVESKTKPFTLPILTLGELSNSRFPVSIDQMYTSPNEIISVQCQNGRCTLDGELQGTTQLQVSGICAFKGEVTAHLHDNDHLYNVTITNLNGSPFDPSEDIPAPLGVPDFQGRVFGIISQRDKHNSPGHNEPANRGHDAVVPTYTAQYTPKLGQIQIGTWQTDDLTVNQPVKFTPVGLNDTEHFNQWVVPRYAGALNLNTNLAPSVAPVFPGERLLFFRSYIPLKGGYGNPAIDCLLPQEWVQHFYQEAAPSMSEVALVRYINPDTGRALFEAKLHRAGFMTVSSNTSAPVVVPANGYFRFDSWVNQFYSLAPMGTGNGRRRVQ

[0277] SEQ ID NO. 12 Norovirus genotype GI.1 (Norovirus Hu / GI.1 / 8McIII / 1973 / USA; Gene ID: KF429770.1, NC_001959.2)

[0278] MMMASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGVLSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR

[0279] SEQ ID NO. 13

[0280] Norovirus genotype GI.3 (Norwalk-like virus NLV / Honolulu / 219 / 1992 / US; GeneID: AF414403)

[0281] MMMASKDAPPNMDGTSGAGQLVPEANTAEPISMEPVAGAATAAATAGQVNMIDPWIMNNYVQAPQGEFTISPNNTPGDILFDLQLGPHLNPFLSHLAQMYNGWVGNMKVKVLLAGNAFTAGKIIISCIPPGFAAQNISIAQATMFPHVIADVRVLEPIEVPLEDVRNVLFHNNDNTPTMRLVCMLYTPLRASGSSSGTDPFVIAGRVLTCPSPDFSLLFLVPPNVEQKTKPFSVPNLPLNTLSNSRVPSLIKSMMVSRDHGQMVQFQNGRVALDGQLQGTTPTSASQLCKIRGSVFHANGGNGYNPTELDGSPYHAFESPAPIGFPDLGECDWHMEASPTTQFDTGDVIKQINVKQESAFAPHLGTIQADGLSDVSVNTNMIAKLGWVSPVSDGHRGNVDPWVIPRYGSTLTEAAQLAPPIYPPGFGEAIVFFMSDFPIAHGTNGLSVPCTIPQEFVTHFVNEQAPTRGEAALLHYLDPDTHRNLGEFKLYPEGFMTCVPNSSGTGPQTLPINGVFVFVSWVSRFYQLKPVGTAGPARRLGIRRS

Claims

1. A recombinant expression vector for producing a norovirus vaccine, wherein, Contains polynucleotides, The aforementioned polynucleotides encode norovirus antigen proteins; and mutant proteins in which three or four amino acid residues in the RNA interaction domain sequence isolated from mammalian lysyl-tRNA synthetase are replaced with other amino acid residues.

2. The recombinant expression vector for producing norovirus vaccine according to claim 1, wherein, The RNA interaction domain described above consists of the amino acid sequences of SEQ ID NO. 2 and SEQ ID NO.

3.

3. The recombinant expression vector for producing norovirus vaccine according to claim 1, wherein, The substituted amino acid residues include at least one of the amino acid residues at positions 19, 23, 24, 30, 31, 38 and 40 of the amino acid sequence of SEQ ID NO.

3.

4. The recombinant expression vector for producing a norovirus vaccine according to claim 1, wherein, The mutant protein of the above RNA interaction domain contains one of the amino acid sequences of SEQ ID NO. 5 to SEQ ID NO.

8.

5. The recombinant expression vector for producing a norovirus vaccine according to claim 1, wherein, The norovirus antigen protein described above contains at least one amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO.

13.

6. A host cell, wherein, Transformation using the expression vector according to any one of claims 1 to 5.

7. The host cell according to claim 5, wherein, The host cell mentioned above is Escherichia coli.

8. A fusion protein for use in a norovirus vaccine, wherein, Include: Norovirus antigen protein; and Mutant proteins in which three or four amino acid residues in the RNA interaction domain sequence isolated from mammalian lysyl-tRNA synthetase are replaced with other amino acid residues.

9. The fusion protein according to claim 8, wherein, The RNA interaction domain is composed of the amino acid sequences of SEQ ID NO. 2 and SEQ ID NO. 3, and the substituted amino acid residues include at least one of the amino acid residues at positions 19, 23, 24, 30, 31, 38 and 40 of the amino acid sequence of SEQ ID NO.

3.

10. The fusion protein according to claim 8, wherein, The mutant protein of the above RNA interaction domain consists of at least one amino acid sequence selected from the group consisting of SEQ ID NO. 5 to SEQ ID NO.

8.

11. The fusion protein according to claim 8, wherein, The norovirus antigen protein described above contains at least one amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO.

13.

12. A virus-like particle, wherein, It is self-assembled from the antigen protein cleaved from the fusion protein of claim 8.

13. A method for producing norovirus-like particles, wherein, include: Step a) Production of a recombinant expression vector for producing a vaccine containing a polynucleotide, wherein the polynucleotide encodes a norovirus antigen protein and a mutant protein in which three or four amino acid residues in the sequence of the RNA interaction domain isolated from mammalian lysyl tRNA synthetase are replaced with other amino acid residues. Step b) involves introducing the expression vector into host cells to produce the transformant; and Step c), after culturing the above-mentioned transformants and inducing the expression of the recombinant fusion protein, the transformants are obtained.

14. The method for producing norovirus-like particles according to claim 13, wherein, The fusion protein prepared above contains tobacco etched virus protein between the norovirus antigen protein and the RNA interaction domain protein. The above production method includes step d) after step c), which uses a tobacco etch virus cleavage enzyme to cleave the RID protein of the prepared fusion protein with the norovirus antigen protein.

15. The method for producing norovirus-like particles according to claim 14, wherein, Following step d) above, including step e), the cleaved antigen protein is purified.

16. The method for producing norovirus-like particles according to claim 15, wherein, Following step e) above, a step is included to allow the purified protein to self-assemble into virus-like particles.

17. The method for producing norovirus-like particles according to claim 13, wherein, The RNA interaction domain is composed of the amino acid sequences of SEQ ID NO. 2 and SEQ ID NO. 3, and the substituted amino acid residues include at least one of the amino acid residues at positions 19, 23, 24, 30, 31, 38 and 40 of the amino acid sequence of SEQ ID NO.

3.

18. The method for producing norovirus-like particles according to claim 13, wherein, The mutant protein of the RNA interaction domain described above contains one amino acid sequence from the amino acid sequences of SEQ ID NO. 5 to SEQ ID NO.

8.

19. The method for producing norovirus-like particles according to claim 13, wherein, The norovirus antigen protein described above contains at least one amino acid sequence selected from the group consisting of SEQ ID NO. 9 to SEQ ID NO. 13.

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