Recombinant expression vector and manufacturing method for virus-like particle-based norovirus polyvalent vaccine production

Abstract

The present invention provides a fusion protein and an expression vector for enhancing the soluble expression of norovirus antigens. More specifically, it provides a combination of diverse genotypes of norovirus antigen proteins and RID mutations that, when norovirus antigens are used as target proteins, fold most efficiently during the formation of recombinant fusion proteins, improve the soluble expression yield of the produced fusion proteins, and enhance the assembly efficacy and homogeneity of norovirus VLPs. Furthermore, by utilizing mutational RIDs, the present invention provides a method for rapidly and in large quantities producing not only GII.4 but also other diverse genotypes of norovirus VLPs in E. coli, thereby enabling the development of VLP-based multivalent vaccines containing a wider range of genotypes.

Description

Technical Field

[0001] The present invention relates to a recombinant expression vector and a method for producing virus-like particles for highly efficient and rapid production of norovirus multivalent vaccines based on virus-like particles in Escherichia coli. More specifically, the present invention relates to a recombinant expression vector capable of highly efficiently and solubly expressing antigen proteins of noroviruses of various genotypes in Escherichia coli and an efficient method for producing norovirus multivalent vaccines using the same.

[0002] This invention was carried out with the support of the research project "Development of Norovirus Enteritis VLP-Type Multivalent Preventive Vaccine" (Project Specific Number: 1465035890, Research Period: October 06, 2020 to December 31, 2023) of the Infectious Disease Prevention and Treatment Technology Development Project supported by the Ministry of Health and Welfare of Korea, and the research project "Development of Dosage Forms for Enhancing the Long-Term Stability of Norovirus VLP-Type Vaccine Candidates" (Project Specific Number: 2023112201, Research Period: July 01, 2021 to December 31, 2023) of the Regional Vaccine R&D Infrastructure Activation Project supported by the Ministry of Education of Korea.

Background Art

[0003] Norovirus, the virus to which the technology of this invention is applied, is one of the most important causative pathogens of acute gastroenteritis and food poisoning. According to the FERG (Foodborne Disease Burden Epidemiology Reference Group) under the WHO, approximately 700 million norovirus infections are reported worldwide every year, resulting in over 200 million infections in children under 5 years old and 50,000 deaths annually. Norovirus infection is mainly transmitted through person-to-person contact, aerosols, and the fecal-oral route of infected individuals, and can also be transmitted through contaminated water and food. Norovirus can infect people of all ages, but it occurs more frequently in infants and the elderly. Disease can develop with very small amounts of infection, the virus is shed for a long period, and remains stable at temperatures below 60°C. Furthermore, due to factors such as persistent antigenic mutation and genetic recombination, it is impossible to acquire long-lasting immunity, allowing for widespread outbreaks, and outbreaks occur in schools, hospitals, nursing homes, and other facilities. The most common symptoms of infection with the pathogen are abdominal pain, low-grade fever, vomiting, and diarrhea. The typical incubation period is 24 to 48 hours, and symptoms vary from person to person, ranging from asymptomatic to fever, vomiting, diarrhea, abdominal pain, and dehydration. In particular, severe dehydration can lead to death in the elderly.

[0004] Norovirus is a membrane-less virus belonging to the Caliciviridae family, with a diameter of approximately 30-40 nm. It consists of a 7.6 kbp single-stranded (+) RNA molecule. Of its three open reading frames (ORFs), ORF2 encodes VP1, the main structural protein that gives norovirus its form, while ORF3 encodes the VP2 protein, although it is not directly involved in structural formation. VP1 is approximately 59 kDa in size, and 90 dimers self-assemble to form a dimer, with all 180 VP1 molecules forming a single viral particle. The VP1 protein consists of two domains: the S domain is involved in structural formation, while the P domain is known to be involved in inducing the actual immune response. The P2 region of the P domain has emerged as a potential vaccine target because it is known to bind to HBGAs (histo-blood group antigens).

[0005] Norovirus is classified into seven genogroups based on the overall amino acid sequence of VP1. GI, GII, and GIV induce human infection, but the most frequently detected genotype in outbreaks is GII.4, accounting for 50-70% of the overall prevalence. GII.4 alters its antigenicity through mutations in the P2 domain HBGA binding site of VP1, and can evade the immune system through diverse genetic recombinations, resulting in global outbreaks of new variants every 2-3 years. In addition to GII.4, there was a rapid outbreak of GII.17 in the Asian region from 2014 to 2015 (Chan et al., Nature communications 2015), and since 2016, GII.3 has been frequently detected (Kuang, et al. Gut Pathog 2019). Globally, the prevalence of GII.3 is increasing, and in South Korea, both GII.3 and GII.2 genotypes have been detected in the last three years. Norovirus is genetically highly diverse, and new variants continue to emerge, so the possibility of a dominant genotype replacing GII.4 cannot be ruled out in the future. Therefore, there is a strong need to develop a multivalent vaccine that provides preventive efficacy against many possible genotypes, including additional genotypes of the GII gene group (Genogroup), which account for more than 90% of the prevalence of norovirus infection.

[0006] Vaccines for preventing viral infections have traditionally been manufactured in the form of live vaccines, which are attenuated viruses, or inactivated vaccines, which are produced after viral culture and purification. However, in the case of norovirus, there is no known culture method that can grow the virus, and developing live and inactivated vaccine formulations is not only difficult, but suitable animal models have also not been developed. As a result, there are currently no commercially available norovirus vaccines. Therefore, vaccine development based on VLP (Virus-Like Particles) technology, which is not constrained by the ability of the virus to grow in host cells, is becoming the mainstream approach.

[0007] Virus-like particles (VLPs) are highly complex and sophisticated structures created by specifically expressing viral structural proteins to exhibit a structure similar in appearance to wild-type viruses. They have a size of 20-100 nm in diameter and can be produced through virion assembly after specifically expressing viral structural proteins with known sequences using various types of recombinant protein expression systems. While possessing the same external appearance and structure as infectious viruses, they lack viral-derived genes and therefore cannot replicate. However, they induce high immunogenicity through high-density and highly ordered antigen expression. Because their structure is similar to wild-type viruses, they have the advantage of stimulating both T-cell and B-cell immune pathways in the body. However, due to their complex structure and the significant differences in characteristics between different viruses, only a few VLPs have actually been approved as commercial vaccines, due to the need for complex manufacturing processes or low productivity.

[0008] Norovirus VLPs can be produced primarily using baculovirus-insect cells, and it is known that VLPs can also be produced in yeast. However, these methods have disadvantages, such as long production periods, the need for expensive production equipment, and high direct manufacturing costs. Considering the large number of deaths and injuries caused by norovirus infection in developing countries, the production of low-cost vaccines through highly efficient and rapid production is essential. To make this possible, the production of VLPs derived from E. coli, which allows for highly efficient and low-cost mass production of viral antigen proteins, is the most preferable. However, in the case of E. coli, although highly efficient expression of VP1 proteins of each norovirus genotype is possible, the solubility of the protein is significantly reduced, and as a result, the efficiency of VLP formation is known to be rather low. Therefore, in order to produce norovirus VLPs derived from E. coli, it is essential to first express a fusion protein with a soluble expression tag to improve the soluble expression rate of the antigen protein.

[0009] In previous research, the inventors confirmed that when an RNA interaction domain (RID) containing ARSNTD (aminoacyl RNA synthetase N-terminal domain) was fused to the N-terminus of the norovirus VP1 protein and expressed, not only was the soluble expression of the VP1 protein increased through the action of the intrinsically disordered peptide structure (IDP) of the RID, but a complete VP1 antigen protein was also efficiently produced through a protein folding induction mechanism of tRNA attached to the RID. The inventors verified that the target protein purified with the fusion protein could rapidly produce a large number of virus-like particles (VLPs) after purification through self-assembly without the need for a separate refolding process (Korean Patent Publication No. 1914779 & Korean Patent Publication No. 2120335).

[0010] However, while the aforementioned invention worked well for GII.4, a VP1 antigen protein with a relatively stable structure, it was observed that its soluble expression rate significantly decreased when applied to other genotypes of norovirus VP1 protein. To solve this, a mutant (hereinafter referred to as m9 RID) was created by substituting all nine positively charged lysine (K) or arginine (R) residues in the entire RID sequence with alanine (A). This resulted in lower tRNA binding efficiency but a mutant that was more advantageous for maintaining the intrinsically disordered structure (IDP). When this mutant was applied, it was found to be very effective in soluble expression of various genotypes of norovirus VP1 protein other than GII.4 compared to when a wild-type RID (wtRID) without mutations was attached (Korean Patent Publication No. 2038876).

[0011] In the case of the aforementioned m9 RID, while soluble expression can be improved through the action of the IDP structure, tRNA binding is eliminated, so the effect of inducing target protein folding is greatly reduced. Although the soluble expression level is high, the specific gravity of soluble aggregates is high, and the specific gravity of target proteins with the appropriate structure is very low, which is a problem. Consequently, in the case of m9 RID, although the soluble expression level is high, the amount of VLP that can be ultimately obtained does not increase proportionally, resulting in the disadvantage of lower final production efficiency.

[0012] To solve the aforementioned problems, the inventors analyzed protein motifs using databases such as Pfam, NCBI-CDD, and PROSITE, as well as the MOTIF tool. Based on the results, they manufactured novel mutant m3 RIDs or m4 RIDs that maintain the function of inducing target protein folding by binding to native RNA, while also maintaining the IDP structure essential for enhanced soluble expression, by replacing three or four lysine (K) or arginine (R) residues in specific sequences of wild-type RIDs with alanine (A).

[0013] The present invention was completed by confirming that when the aforementioned mutant RID was attached to the N-terminus of various genotypes of norovirus VP1 protein and expressed, not only was soluble expression greatly improved, but the efficiency of virus-like particle (VLP) production through self-assembly was also greatly improved.

[0014] By utilizing a novel mutant RID (m3 or m4 RID) that possesses both improved soluble expression efficiency, which is the core of this invention, and the ability to induce target protein folding by RNA adsorbed to the binding site, we have demonstrated the inventiveness of this technology by providing a method for rapidly producing not only GII.4 but also other diverse genotypes of norovirus VLP in E. coli on a large scale.

[0015] In particular, the significance of this invention lies in the fact that by enabling the development of VLP-based multivalent vaccines containing a wider range of genotypes, it becomes possible to provide an appropriate solution for preparing for the threat of novel and acute epidemic pathogens caused by persistent genetic mutations in the future. [Overview of the project] [Problems that the invention aims to solve]

[0016] The present invention aims to provide a fusion protein and virus-like particles that can be used for the prevention or treatment of infections such as norovirus infections.

[0017] The main objective of this invention is to present a mutant RID gene combination that can most efficiently express a soluble fusion protein when a norovirus antigen protein is used as the target protein. [Means for solving the problem]

[0018] To solve the aforementioned technical problems, the present invention provides a recombinant expression vector for norovirus vaccine production, 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 lysyl-tRNA synthetase (hLysRS, human lysyl-tRNA synthetase) are replaced with other amino acid residues.

[0019] In the present invention, it is preferable that the RID consists of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3.

[0020] Preferably, the amino acid residue to be substituted in the RID sequence of the present invention includes at least one of the residues at positions 19, 23, 24, 30, 31, 38, and 40 in the amino acid sequence of SEQ ID NO: 3. More preferably, SEQ ID NO: 3 may include at least one mutation from K19A, K23A, R24A, K30A, K31A, K38A, and K40A. For example, the amino acids at positions 23, 30, and 31 in the amino acid sequence of SEQ ID NO: 3 may be substituted, or the amino acids at positions 19, 38, and 40 may be substituted. According to one embodiment of the present invention, the mutant protein of the RID may include any one of the amino acid sequences from SEQ ID NO: 5 to SEQ ID NO: 8.

[0021] The norovirus antigen protein of the present invention is not limited in its genotype, but is preferably an antigen protein that contains at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 13.

[0022] The present invention also provides host cells transformed with the expression vector, preferably Escherichia coli (E. coli).

[0023] Another embodiment of the present invention provides a fusion protein for a norovirus vaccine comprising a norovirus antigen protein; and a mutant protein in which three or four amino acid residues of the RNA interacting domain (RID) sequence isolated from mammalian lysyl tRNA synthetase (hLysRS, human lysyl tRNA synthetase) are replaced with other amino acid residues. In the fusion protein of the present invention, at least one linker protein may be located between the norovirus antigen protein and the mutant RID protein.

[0024] The present invention also provides virus-like particles formed by self-assembly of an antigen protein in which RID is cleaved in the fusion protein. The virus-like particles (VLPs) of the present invention may be formed with a size of 30 to 40 nm.

[0025] According to another embodiment of the present invention, there is provided a method for the soluble production of a norovirus antigen protein (or a method for the production of norovirus-like particles), comprising: (a) producing a recombinant expression vector for vaccine production, which contains a polynucleotide encoding (a) a norovirus antigen protein; and (b) a mutant protein in which three or four amino acid residues of the sequence of RID (RNA interacting domain) separated from mammalian LysRS (mammalian lysyl tRNA synthetase) are substituted with other amino acid residues; (b) introducing the expression vector into a host cell to produce a transformant; and (c) culturing the transformant, inducing the expression of the recombinant fusion protein, and then obtaining the same.

[0026] In the above production method, the fusion protein may contain a TEV (tobacco etch virus) protein between the norovirus antigen protein (the target protein of the present invention) and the RID protein, and after step (c), it may include (d) a step of cleaving between the RID protein and the norovirus antigen protein of the produced fusion protein using a TEV cleavage enzyme.

[0027] Also, after step (d), it may include (e) a step of purifying the cleaved antigen protein; and after step (e), a step of self-assembly of the purified protein to form virus-like particles.

[0028] In the production method of the present invention, the RID consists of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3, and among the amino acid residues to be substituted, at least one of the 19th, 23rd, 24th, 30th, 31st, 38th, and 40th amino acid residues in the amino acid sequence of SEQ ID NO: 3 may be included.

[0029] It is most preferable that the mutant protein of the RID contains any one of the amino acid sequences of SEQ ID NOs: 5 to 8.

[0030] In the production method, the norovirus antigen protein may contain at least any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 9 to 13.

Advantages of the Invention

[0031] A peptide containing the amino acid sequence encoding the mutant RID protein according to the present invention can significantly enhance the soluble expression of the target protein, the norovirus antigen protein, when producing a recombinant protein (fusion protein) in Escherichia coli.

[0032] In addition, when forming a recombinant protein using not only GII.4 but also norovirus antigen proteins of various genotypes as target proteins, the present invention provides a mutant form of RID in which folding occurs most efficiently and the production yield of the final virus-like particles is improved.

[0033] In addition, the present invention enables the development of VLP-based multivalent vaccines containing more genotypes by providing a method for mass-producing norovirus VLPs of various genotypes other than GII.4 in Escherichia coli quickly and in large quantities by utilizing mutant RID.

Brief Description of the Drawings

[0034] [Figure 1a]Figure 1 shows a schematic diagram of a recombinant expression vector for norovirus VP1 protein production according to an example of the present invention, and the expression results of the highly efficient soluble protein depending on the expression level and the presence or absence of mutations in RID, the solubility-enhancing fusion partner. Figure 1a is a schematic diagram showing the structure of a pET9a-based norovirus VP1 protein recombinant expression vector containing RID and mutant RID. [Figure 1b] Figure 1 shows a schematic diagram of a recombinant expression vector for norovirus VP1 protein production according to an example of the present invention, and the expression levels and the results of highly efficient soluble protein expression depending on the presence or absence of mutations in RID, the solubility-enhancing fusion partner. Figure 1b shows the results of classifying the presence or absence of soluble expression of norovirus VP1 protein bound to RID according to the norovirus genotype and confirming it by SDS-PAGE. [Figure 1c] Figure 1 shows a schematic diagram of a recombinant expression vector for norovirus VP1 protein production according to an example of the present invention, and the expression results of the highly efficient soluble protein depending on the expression level and the presence or absence of mutations in the RID, which is the solubility-enhancing fusion partner. Figure 1c shows data comparing the degree of soluble expression of norovirus VP1 protein bound to RID or mutant RID (mRID) produced according to one example of the present invention. [Figure 1d] Figure 1 shows a schematic diagram of a recombinant expression vector for norovirus VP1 protein production according to an example of the present invention, and the results of highly efficient soluble protein expression depending on the expression level and the presence or absence of mutations in RID, the solubility-enhancing fusion partner. Figure 1d shows the results of confirming the presence or absence of highly efficient soluble expression of norovirus GII.17 VP1 protein bound to RID and mutant RID using the expression vector produced according to the present invention, as confirmed by SDS-PAGE. [Figure 1e]Figure 1 shows a schematic diagram of a recombinant expression vector for norovirus VP1 protein production according to an example of the present invention, and the expression results of highly efficient soluble protein depending on the expression level and the presence or absence of mutations in the RID, which is the solubility-enhancing fusion partner. Figure 1e shows the overall shape of VLPs (Virus-like particles) based on norovirus GII.17 VP1 protein, purified after expression containing four types of RID wild-type and mutant types, as confirmed by transmission electron microscopy (TEM). [Figure 2a] Figure 2a shows the results of confirming the effect of the presence or absence of the m3c mutation in RID on the soluble expression of the norovirus GII.17 VP1 fusion protein according to an example of the present invention. Unlike the norovirus GII.4 VP1 protein, when RID is fused to the N-terminus of the norovirus GII.17 VP1 protein, it is mostly expressed insolublely, while when m3c is fused to the N-terminus and expressed, it is mostly expressed solublely, as confirmed by SDS-PAGE. [Figure 2b] Figure 2b shows the protein separation pattern confirmed by SDS-PAGE after purification by nickel affinity chromatography of norovirus GII.17 VP1 fusion protein containing m3c, one of the mutant RIDs expressed in the examples of the present invention. [Figure 2c] Figure 2c shows the results of confirming the entire process, from cleavage of the RID(EE domain+m3c)-norovirus GII.17 VP1 fusion protein eluted by nickel affinity chromatography using a TEV enzyme to acquisition of the flow-through (FT) fraction eluted by subsequent nickel affinity chromatography, using SDS-PAGE. [Figure 2d] Figure 2d shows the purification process of norovirus GII.17 VP1 protein eluted by nickel affinity chromatography and purified via ion resin exchange chromatography, as confirmed by SDS-PAGE. [Figure 2e]Figure 2e shows the diameter distribution of virus-like particles (VLPs) produced after the self-assembly step of purified norovirus GII.17 VP1 protein, analyzed via DLS (Dynamic Light Scattering). [Figure 2f] Figure 2f shows the overall shape of the manufactured norovirus GII.17 virus-like particles as confirmed by transmission electron microscopy (TEM). [Figure 2g] Figure 2g is a schematic diagram showing the binding configuration for HBGA binding affinity analysis according to one embodiment of the present invention. [Figure 2h] Figure 2h shows the results of an analysis of the degree to which GII.4, GII.17, and GII.3 VLP proteins bind to Blood type B(tri), Blood type B(tri), and Le a(H type1), respectively, with the binding affinity of each VLP expressed in EC50. [Figure 3a] Figure 3a is a schematic diagram showing the experimental procedure performed to measure the specific antibody-inducing ability of GII.17 virus-like particles (VLPs) produced according to one embodiment of the present invention. [Figure 3ba] Figure 3ba shows the results of measuring the degree of specific antibody induction at different dose levels in serum collected after a single administration of virus-like particles (NoV GII.17 VLP) according to the present invention, using enzyme-linked immunosorbent assay (ELISA). [Figure 3bb] Figure 3bb shows the results of measuring the degree of specific antibody induction at different dose levels in serum collected after two administrations of virus-like particles (NoV GII.17 VLP) according to the present invention, using enzyme-linked immunosorbent assay (ELISA). [Figure 3bc]Figure 3bc shows the results of measuring the degree of specific antibody induction at different dose levels in serum collected after three administrations of virus-like particles (NoV GII.17 VLP) according to the present invention, using enzyme-linked immunosorbent assay (ELISA). [Figure 3c] Figure 3c is a schematic diagram showing the binding configuration for analyzing the inhibitory efficacy of the immunoserum on binding between the GII.17 VLP antigen (a VLP consisting of the antigen protein purified after the RID (EE domain + m3c)-NoV GII.17 VP1 fusion protein of the present invention is cleaved with the RID, Nov VLP in Figure 3c) and HBGA. [Figure 3d] Figure 3d shows the results of confirming the differences in HBGA binding inhibitory ability among primary, secondary, and tertiary vaccination groups after inoculation with different doses of norovirus GII.17 VLP according to one embodiment of the present invention. [Figure 4a] Figure 4a shows the results of confirming the effect of the presence or absence of RID mutations on the soluble expression of norovirus GII.3 VP1 protein according to the embodiment of the present invention. When the protein had RID fused to its N-terminus, it showed mostly insoluble expression, whereas when RID (EE domain + m3c) was fused to its N-terminus and expressed, it was confirmed via SDS-PAGE that most of it was expressed solublely. [Figure 4b] Figure 4b shows the protein separation pattern confirmed by SDS-PAGE after purification by nickel affinity chromatography of norovirus GII.3 VP1 protein, including the m3c mutant RID expressed in the examples of the present invention. [Figure 4c] Figure 4c shows the results of confirming the entire process, from cleavage of the RID(EE domain+m3c)-norovirus GII.3 VP1 fusion protein eluted by nickel affinity chromatography using TEV enzyme to acquisition of the flow-through (FT) fraction eluted by subsequent nickel affinity chromatography, using SDS-PAGE. [Figure 4d]Figure 4d shows the purification process of norovirus GII.3 VP1 protein eluted by nickel affinity chromatography and purified via ion resin exchange chromatography, as confirmed by SDS-PAGE. [Figure 4e] Figure 4e shows the diameter distribution of virus-like particles (VLPs) produced after a self-assembly step using purified norovirus GII.3 protein, analyzed via DLS (Dynamic Light Scattering). [Figure 4f] Figure 4e shows the diameter distribution of virus-like particles (VLPs) produced after a self-assembly step using purified norovirus GII.3 protein, analyzed via dynamic light scattering (DLS). Figure 4f shows the overall shape of the produced norovirus GII.3 virus-like particles, confirmed by transmission electron microscopy (TEM). [Figure 5a] Figure 5a is a schematic diagram showing the experimental procedure performed to measure the specific antibody-inducing ability of GII.3 virus-like particles (VLPs) produced according to one embodiment of the present invention. [Figure 5ba] Figure 5ba shows the results of measuring the degree of specific antibody induction at different dose levels in serum collected after a single administration of virus-like particles (NoV GII.3 VLP) according to the present invention, using enzyme-linked immunosorbent assay (ELISA). [Figure 5bb] Figure 5bb shows the results of measuring the degree of specific antibody induction at different dose levels in serum collected after two administrations of virus-like particles (NoV GII.3 VLP) according to the present invention, using enzyme-linked immunosorbent assay (ELISA). [Figure 5bc]Figure 5bc shows the results of measuring the degree of specific antibody induction at different dose levels in serum collected after three administrations of virus-like particles (NoV GII.3 VLP) according to the present invention, using enzyme-linked immunosorbent assay (ELISA). [Figure 5c] Figure 5c shows the results of confirming the differences in HBGA binding inhibitory ability among primary, secondary, and tertiary vaccination groups after inoculation with different doses of norovirus GII.3 VLP according to one embodiment of the present invention. [Figure 6a] Figure 6a shows the results of examining the effect of expression levels and the presence or absence of m3c mutations in RID, a fusion partner that enhances solubility, on the soluble expression of proteins fused with norovirus GI.1 VP1 protein. When RID was fused to the N-terminus, the majority of the expression was insoluble, whereas when RID (EE domain + m3c) was fused to the N-terminus and expressed, the majority of the expression was soluble, as confirmed by SDS-PAGE. [Figure 6b] Figure 6b shows the results of confirming the effect of the presence or absence of mutations in the RID (particularly the SE domain of the RID) on the soluble expression of the norovirus GI.3 VP1 fusion protein according to the embodiment of the present invention. When the protein was fused with RID at its N-terminus, most of it showed an insoluble expression pattern, whereas when RID (EE domain + m3c) was fused to its N-terminus and expressed, most of it was expressed solublely, as confirmed by SDSPAGE. [Figure 6c] Figure 6c shows the results of confirming the effect of the presence or absence of m3c mutations in RID, the expression level and solubility-enhancing fusion partner, on the soluble expression of the norovirus GII.2 VP1 protein and target protein fusion protein according to the embodiment of the present invention. When the protein was fused with RID at its N-terminus, it showed mostly insoluble expression, whereas when RID (EE domain + m3c) was fused to its N-terminus and expressed, it was confirmed via SDS-PAGE that most of it was expressed solublely. [Figure 6da]Figure 6da shows the size distribution (upper panel of Figure 6da) and particle formation of norovirus GI.1 VLP particles produced solely from the norovirus GI.1 VP1 protein that was finally purified after fusion protein expression with RID (EE domain + m3c) according to the present invention, as confirmed by DLS (Dynamic Light Scattering) and transmission electron microscopy (TEM), respectively. [Figure 6db] Figure 6db shows the size distribution (upper panel of Figure 6db) and particle formation of norovirus GI.1 VLP particles produced solely from the norovirus GI.3 VP1 protein, which was finally purified after fusion protein expression with RID (EE domain + m3c) according to the present invention. [Figure 6dc] Figure 6dc shows the size distribution (upper panel of Figure 6dc) and particle formation of norovirus GII.2 VLP particles produced solely from the norovirus GII.2 VP1 protein, which was purified after the expression of a fusion protein with RID (EE domain + m3c) according to the present invention. [Modes for carrying out the invention]

[0035] The present invention will be described in detail below. The advantages and features of the present invention will become clear when referred to the embodiments described below that achieve them. However, the present invention is not limited to the embodiments listed below and can be embodied in a variety of different forms, and these embodiments are provided merely to complete the listing of the present invention and to fully inform those who are ordinary skill in the art to which the present invention pertains, and the present invention is defined only by the scope of the claims. The same reference numerals throughout the specification refer to the same components.

[0036] Unless otherwise specified, all terms used herein (including technical and scientific terms) should be used in a sense that is commonly understood by a person of ordinary skill in the art to which the present invention pertains. Furthermore, terms defined in commonly used dictionaries should not be interpreted ideally or excessively unless explicitly defined otherwise. Terms used herein are for illustrative purposes only and are not intended to limit the present invention. In this specification, the singular form includes the plural form unless otherwise specified in the text.

[0037] As used herein, the term "target protein" refers to a protein that a person skilled in the art would like to produce in large quantities. The recombinant expression vector of the present invention contains a polynucleotide encoding the target protein and can be expressed in a host cell.

[0038] According to one embodiment of the present invention, the target protein includes a viral antigen protein, an antibody, a cell receptor, an enzyme, a structural protein, serum, and a cell protein. According to one preferred embodiment of the present invention, the viral antigen protein is a norovirus antigen protein.

[0039] The norovirus antigen protein of the present invention may include GI and GII types of norovirus, 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 consist of the amino acid sequences shown in Sequence IDs 9 to 13, but is not limited thereto.

[0040] As used herein, the terms “RID,” “RNA interacting domain,” and “N terminal appended RNA binding domain of Lysyl tRNA synthetase (ARSNTD)” refer to the unique N-terminal extension of mammalian LysRS involved in the interaction between RNA and other proteins.

[0041] According to one embodiment of the present invention, the RID protein can be used as a fusion partner to increase the expression level or solubility (or water-soluble properties) of the target protein.

[0042] According to one embodiment of the present invention, the RID is the amino acid sequence of the N-terminal domain (hRID, hLysRS N-terminal appended RNA interacting domain) isolated from mammalian lysyl tRNA synthetase (hLysRS, human lysyl tRNA synthetase) (wild type), and the mutant RID includes a mutant sequence in which one or more positively charged amino acid residues in the RID sequence are replaced with other amino acid residues.

[0043] According to a preferred embodiment of the present invention, the RID protein according to the present invention comprises an EE domain (SEQ ID NO: 2, MSAVKAA) used to increase the expression level of a target protein and an SE domain (SEQ ID NO: 3) used to enhance soluble expression through folding induction of the target protein.

[0044] The RID protein used in the present invention may be mutated. As used herein, the term "mutation" means that one or more residues in an amino acid sequence are replaced by other amino acid residues, some residues in an amino acid sequence are deleted, or a specific amino acid residue is inserted. In the present invention, "mutation" is used interchangeably with "point mutation" or "site-directed mutation" and refers to a mutation in which an amino acid residue at a specific position is replaced by another amino acid residue.

[0045] 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.

[0046] According to one embodiment of the present invention, the mutation in RID preferably involves the substitution of 3 to 4 amino acid residues on the SE domain. For example, 3 to 4 amino acid residues may be substituted in the amino acid sequence of SEQ ID NO: 3 (the SE domain of RID in the present invention). According to a preferred embodiment of the present invention, in the amino acid sequence of the SE domain of the present invention (SEQ ID NO: 3), the substituted amino acid residues may include at least one of the 19th, 23rd, 24th, 30th, 31st, 38th, and 40th amino acids. For example, in the amino acid sequence of SEQ ID NO: 3, the 23rd, 30th, and 31st amino acids may be substituted, or the 19th, 38th, and 40th amino acids may be substituted. Preferably, the substitution targets 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 of the amino acid residues at positions 23, 24, 30, and 31 (e.g., K23, R24, K30, and K31) may be substituted. That is, according to one embodiment of the present invention, the amino acid sequence of RID mutated by the present invention may further include one of the sequences from SEQ ID NOs: 5 to 8 in addition to SEQ ID NO: 2.

[0047] The names of the sequences used in this invention are organized as shown in Table 1 below.

[0048] [Table 1]

[0049] In the present invention, the EE domain may include the amino acid sequence of SEQ ID NO: 2 or a portion thereof.

[0050] According to one embodiment of the present invention, when the norovirus antigen protein is the target protein during the production of the fusion protein of the present invention, the solubility (or water-soluble water) of the fusion protein was higher when a mutated RID protein (a protein consisting of one amino acid sequence from SEQ ID NOs. 4 to 8 and the amino acid sequence of SEQ ID NO. 2) was bound to the fusion protein than when a non-mutated RID protein was bound (see Figure 1c). Furthermore, when the norovirus antigen protein was bound to the m3 or m4 protein, which has 3 or 4 amino acid residue substitutions, rather than the m9 protein with 9 mutations in the SE domain, protein folding occurred more easily (see Figure 1e). When the norovirus antigen protein was bound to the RID containing m3 or m4 with 3 or 4 amino acid residue substitutions in the SE domain sequence, an effect of increased soluble expression yield was observed, and the efficiency of VLP formation was improved through more accurate folding induction.

[0051] As used herein, the term "expression vector" refers to a linear or round DNA molecule consisting of a fragment encoding a target protein operably ligated to an additional fragment provided for transcription of the expression vector. Such additional fragments include a promoter and a termination code sequence. The expression vector also includes one or more replication start sites, one or more selection markers, etc. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both. As used herein, the term "operably ligated" indicates that the fragments are arranged to act so as to initiate transcription at the promoter and proceed through the encoding sequence to the termination code.

[0052] In the expression vector according to the present invention, the expression vector may be a plasmid, a viral vector, a phage particle, or a genome insert. The expression vector may be transformed into a host cell and then replicated independently of the host cell's genome, or integrated into the host cell's genome.

[0053] According to one embodiment of the present invention, the polynucleotide may further encode a "linker," and at least one linker protein may be located between the target protein and the RID (RNA interacting domain) protein, respectively.

[0054] According to one preferred embodiment of the present invention, the fusion protein sequence of the present invention, particularly the sequence of the target protein, may include a TEV-specific cleavage sequence (TEV-specific cleavage site) for cleaving the RID.

[0055] In the recombinant expression vector for vaccine production of the present invention, the protein-cleaving enzyme may be TEV.

[0056] The present invention also provides host cells transformed with the expression vector.

[0057] As used herein, the terms "transformation" or "introduction" mean introducing DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through completion of chromosome integration. Methods for transforming an expression vector according to the present invention may include, but are not limited to, electroporation, calcium phosphate (CaPO4), calcium chloride (CaCl2), microinjection, polyethylene glycol (PEG), DEAE-dextran, cation liposome, or lithium acetate-DMSO.

[0058] The present invention also provides a fusion protein comprising a target protein and a RID protein, wherein the RID (RNA interacting domain) protein may include a mutant RID in which three (m3) to four (m4) amino acid residues in the SE domain are substituted.

[0059] As used herein, the term "fusion protein" refers to a protein in which another protein is ligated to the N-terminus or C-terminus of a target protein sequence, or to which another amino acid sequence is added. In the present invention, the fusion protein may also be a vaccine produced by the recombinant expression vector of the present invention. According to one embodiment of the present invention, the fusion protein of the present invention is a protein in which a mutant RID is fused with a target protein (norovirus antigen protein).

[0060] According to one embodiment of the present invention, a fusion protein can be formed using the recombinant expression vector, and the fusion protein may be used in a vaccine either on its own or after any additional steps.

[0061] The present invention also provides virus-like particles (VLPs) formed by combining multiple antigen proteins in which the RID of the fusion protein has been cleaved. The virus-like particles according to the present invention may be formed from purified antigen proteins (e.g., norovirus VP1 antigen protein) in which the mutant RID of the fusion protein is cleaved during the formation process.

[0062] The present invention will be described in more detail below based on examples.

[0063] It will be obvious to those of ordinary skill in the art that these embodiments are for illustrative purposes only and should not be interpreted as limiting the scope of the invention.

[0064] [Example 1] Production of norovirus fusion proteins containing mutant RIDs <Example 1-1> To create virus-like particles composed of the norovirus GII.17 VP1 protein, the VP1 gene (Norovirus Hu / GII.17 / HKG / 2014 / CUHK-NS-405, NCBI access number: KP902566.1) derived from the norovirus GII.17 protein was used. VP1, the norovirus antigen, consists of 539 amino acids, and its protein molecular weight is approximately 59.2 kDa. Based on the aforementioned protein sequence, the synthesized gene sequence was produced after codon optimization in E. coli (SEQ ID NO: 9). (Gene synthesis was commissioned to Bionics Co., Ltd.)

[0065] In this example, RID EE domain peptide (MSAVKAA (SEQ ID NO: 2)) and RID SE domain peptide (SEQ ID NO: 3), which have the effect of increasing expression level and solubility at the N-terminus of the norovirus GII.17 VP1 protein, were used. In addition, in order to confirm the effect of RID mutations, mutant RID SE domain peptides that are more than 50% similar to SEQ ID NO: 3 were used instead of the RID SE domain peptide. These include mutant RID SE domain peptides containing K19A, K38A, and K40A mutations (SEQ ID NO: 5), mutant RID SE domain peptides containing K23A, K30A, and K31A mutations (SEQ ID NO: 7), or nine mutant RID SE domain peptides (SEQ ID NO: 4), respectively. In this example, recombinant proteins fused with each mutant SE domain were produced and their soluble expression patterns were verified.

[0066] Furthermore, while ENLYFQG / S is known as the TEV cleavage site sequence, to achieve higher efficiency, a peptide with the amino acid sequence (GaSb)n (a≧1, b≧1, n≧1) was devised and inserted between the TEV cleavage sequence (ENLYFQ) and the target protein (norovirus antigen protein) sequence. A schematic diagram of the fusion protein expression vector produced in this example is shown in Figure 1a.

[0067] The gene sequence described above was inserted using NdeI and BamHI, which are cleavage enzymes present in the MCS of the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 1) expression vector. The sequence of the final recombinant expression vector produced is as follows:

[0068] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GII.17 VP1 [(Sequence ID 2)-(Sequence ID 3)-GT-6xHIS-TEV-GS-(Sequence ID 9)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAP NGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKILFAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSV ESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQCQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQ FQPTKFTPVGVNDDDDGHPFRQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0069] The aforementioned manufactured composite was named "RID-NoV GII.17 VP1".

[0070] MSAVKAA-m3a-GT-6xHIS-TEV-GS-NoV GII.17 VP1 [(Sequence ID 2)-(Sequence ID 5)-GT-6xHIS-TEV-GS-(Sequence ID 9)] :MSAVKAA-AAVQAAEVKVDGSEPKLSANELKRRLKAEKKVAEKEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKIL FAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQ CQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPF RQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0071] The fabricated complex was named "RID(EE domain+m3a)-NoV GII.17 VP1".

[0072] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GII.17 VP1 [(Sequence ID 2)-(Sequence ID 7)-GT-6xHIS-TEV-GS-(Sequence ID 9)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKIL FAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQ CQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPF RQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0073] The fabricated complex was named "RID(EE domain+m3c)-NoV GII.17 VP1".

[0074] MSAVKAA-m9 RID-GT-6xHIS-TEV-GS-NoV GII.17 VP1 [(Sequence ID 2)-(Sequence ID 4)-GT-6xHIS-TEV-GS-Sequence ID 9)] :MSAVKAA-AAVQAAEVKVDGSEPKLSANELAARLAAEAAVAEEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEGNNETLPLEPVAGAAIAAPVTGQNNIIDPWIRTNFVQAPNGEFTVSPRNSPGEILLNLELGPDLNPYLAHLSRMYNGYAGGVEVQVLLAGNAFTAGKIL FAAVPPNFPVEFLSPAQITMLPHLIVDVRTLEPIMIPLPDVRNTFFHYSNQPNSRMRLVAMLYTPLRSNGSGDDVFTVSCRVLTRPTPDFEFTYLVPPSVESKTKPFSLPILTLSELTNSRFPVPIDSLFTAQNNVLQVQ CQNGRCTLDGELQGTTQLLPSGICAFRGRVTAQINQRDRWHMQLQNLNGTTYDPTDDVPAPLGTPDFKGVVFGMVSQRNVGNDAPGSTRAQQAWVSTYSPQFVPKLGSVNLRISDNDDFQFQPTKFTPVGVNDDDDGHPF RQWELPNYSGELTLNMNLAPPVAPNFPGEQLLFFRSFVPCSGGYNQGIIDCLIPQEWIQHFYQESAPSQSDVALIRYVNPDTGRTLFEAKLHRSGYITVAHSGDYPLVVPANGHFRFDSWVNQFYSLAPMGTGNGRRRAQ

[0075] The fabricated complex was named "RID(EE domain+m9)-NoV GII.17 VP1".

[0076] <Example 1-2> The expression vector prepared in Example 1-1 was used to transform 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 at 250 rpm for 5-7 hours. After that, 1 ml was transferred to 10 ml for a 10-fold dilution, and the cells were cultured again. After about 2-4 hours, when the O.D. 600 reached 0.5-0.7, 0.4 mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17-19 hours (maximum 21 hours). The culture medium was collected by centrifugation to obtain only the precipitated E. coli, which was then stored frozen at -80°C. 3 ml of culture medium was used to prepare the harvested E. coli product. 0.3 ml of lysis buffer solution (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole) was added. After ultrasonic grinding, the total lysate (T) was separated into precipitant (P) and soluble lysate (S) by centrifugation. Analysis by SDS-PAGE confirmed that, as shown in Figure 1d, the norovirus GII.17 VP1 protein with RID added to the N-terminus showed high-efficiency expression, but was mostly insoluble. In contrast, the norovirus GII.17 VP1 protein with mutant RID added to the N-terminus showed high-efficiency soluble expression.

[0077] <Examples 1-3> Using the same culture method as in Examples 1-2, in which highly efficient soluble expression was confirmed, 500 ml of E. coli was cultured and expression was induced, and the resulting cells 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 resuspended cells were pulverized using an ultrasonic cell disruptor. The lysed cells were centrifuged at 13500 rpm for 10 minutes at 4°C, and the supernatant was taken and subjected to Ni+ affinity chromatography using AKTA (GE Healthcare). First, a high-performance Ni Sepharose (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), and then the cell lysate supernatant was loaded onto the equilibrated column at a flow rate of 3-5 ml / min. Norovirus GII.17 VP1 protein was eluted and acquired by increasing injection in a linear concentration gradient in the imidazole concentration range of 10 mM to 300 mM using buffer B buffer [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole] containing buffer A and 300 mM imidazole.

[0078] To confirm the separation pattern of the target protein through SDS-PAGE, the gel was analyzed using the Image Lab (Ver. 6.1) program. After specifying the overall lysate band as the reference band using the program's Quantity tool, the relative quantity value of the supernatant was taken, and the degree of soluble protein expression of each construct was compared and analyzed. As a result, as shown in Figure 1c, it was confirmed that the soluble protein expression rate and the protein yield after purification of the fusion protein containing m3c among the three mutations of the RID were significantly higher than those of the case containing m3a.

[0079] <Examples 1-4> Confirmation of VLP formation based on the type of norovirus antigen-protein binding fusion protein. The external shape of virus-like particles (VLPs) composed of purified norovirus GII.17 VP1 protein was observed using an electron microscope. VLPs composed of norovirus VP1 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 purified using a transmission electron microscope (TEM (120kV); Talos L120C, FEI, Czech). The formation of assembled norovirus VP1 protein VLPs was then confirmed. As shown in Figure 1e, the observed VLPs formed spherical VLPs with diameters between 30 and 40 nm, corresponding to the diameter of norovirus particles. In particular, norovirus GII.17 VP1 protein fused with mutant RIDs containing m3c was found to efficiently form homogeneous VLPs of nearly similar size. On the other hand, while VLP formation was observed with other mutant RIDs, the efficiency was low, and the VLPs contained many intermediates and impurities.

[0080] Through the results of Example 1, we confirmed that when the norovirus VP1 antigen protein containing the mutated RID with a conserved RNA binding site according to the present invention is expressed, not only is an exceptionally high level of expression achieved, but proper protein folding is induced, resulting in higher solubility and superior efficiency and homogeneity of VLP assembly using the final purified VP1 protein.

[0081] [Example 2] Production of Norovirus GII.17 VP1 Virus-like Particles (VLPs) using an Expression Level and Soluble Enhancement Partner (RID) at the N-terminus <Example 2-1> The two expression vectors from Example 1-1 (expression vector for RID-NoV GII.17 VP1 and expression vector for RID(EE domain+m3c)-NoV GII.17 VP1) were used to transform 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 at 250 rpm for 5-7 hours, then 1 ml was transferred to 10 ml for a 10-fold dilution, and further cultures were performed. After approximately 2-4 hours, when the O.D. 600 reached 0.5-0.7, 0.4 mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the culture was incubated at 16°C for 17-19 hours (maximum 21 hours). The culture medium was collected by centrifugation to obtain only the precipitated E. coli, which was then stored frozen at -80°C. 0.3 ml of lysis buffer solution (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 culture medium containing the corresponding E. coli harvest. After ultrasonic grinding, the total lysate (T) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) by centrifugation. Analysis by SDS-PAGE confirmed that the norovirus GII.17 VP1 protein with a mutant RID added to the N-terminus exhibited highly efficient soluble expression, as shown in Figure 2a.

[0082] <Example 2-2> Using the same culture method as in Examples 1-2, in which highly efficient soluble expression was confirmed, 500 ml of E. coli was cultured and expression was induced, and the resulting cells 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 resuspended cells were pulverized using an ultrasonic cell disruptor. The lysed cells were centrifuged at 13500 rpm for 10 minutes at 4°C, and the supernatant was taken and subjected to Ni+ affinity chromatography using AKTA (GE Healthcare). First, a high-performance Ni Sepharose (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), and then the cell lysate supernatant was loaded onto the equilibrated column at a flow rate of 3-5 ml / min. Norovirus GII.17 VP1 protein was eluted and acquired by increasing injection in a linear concentration gradient in the range of 10 mM to 300 mM imidazole using buffer B buffer [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole] containing buffer A and 300 mM imidazole.

[0083] After confirming the separation pattern of the target protein in the eluted material via SDS-PAGE, the eluted material from that fraction was dialyzed overnight (12-16 hours) at 4°C in a dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol], and the bond between the N-terminal RID and the norovirus antigen protein VP1 was cleaved using a TEV cleavage enzyme.

[0084] To separate and purify the target protein from which the N-terminal fusion partner RID and other components have been removed, a high-performance Ni Sepharose (Cytiva) column was equilibrated with the same solution A buffer as the dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% 2-mercaptoethanol], and then loaded onto the equilibrated column at a flow rate of 3-5 ml / min. The NoV GII.17 target protein from which the N-terminal fusion partner RID and other components have been removed was recovered in a form that eluted without being adsorbed onto the column.

[0085] 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 A (50mM Tris-HCl (pH 8.5), 10mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol), and then loaded at a flow rate of 1 ml / min. The target protein was eluted by NaCl injection with a linear concentration gradient of 10mM to 1M through buffers AA and BB (50mM Tris-HCl (pH 8.5), 1M NaCl, 5% glycerol, 0.1% β-mercaptoethanol), and then collected in 1 ml fractions. The purification characteristics and purity are shown in Figures 2b, 2c, and 2d (see Figure 2). The concentration of the final purified protein was quantified using BSA (Amresco, Solon, OH, USA). As can be seen in Figure 2, the RID protein bound within the fusion protein was separated, resulting in successful purification of the target protein.

[0086] <Example 2-3> The norovirus VP1 protein purified in Example 2-2 was stored at 4°C in the final assembly buffer [100 mM potassium phosphate pH 5.8, 150 mM NaCl, 10% glycerol] containing 10% glycerol, with the buffer being changed overnight (12-16 hours). The purified norovirus VP1 protein was assembled in VLP form through a series of processes.

[0087] <Example 2-4> To confirm the overall diameter distribution of virus-like particles (VLPs) assembled from the norovirus VP1 protein purified in Examples 2-3, analysis was performed via dynamic light scattering (DLS). The analysis of the complex was performed using DLS (Particular Systems, Zetasizer Nano Family) with 1 mL of sample placed in a cuvette at a temperature of 16°C. The dynamic light scattering analysis of the cells was confirmed using images, including the total intensity and mass of each region. As shown in Figure 2e, it was confirmed that particles were formed with a size distribution of 30-40 nm in diameter at the hydrodynamic radius of the DLS. (DLS values ​​may be affected by interference depending on the wavelength of light.)

[0088] <Example 2-5> The external shape 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 imaged using a transmission electron microscope (TEM, 120kV; Talos L120C, FEI, Czech). As shown in Figure 2f, it was confirmed that the norovirus VP1 protein assembled after purification formed VLPs. The diameter of the observed VLPs was confirmed to be between 30 and 40 nm, the same as the diameter of norovirus particles, confirming the formation of highly homogeneous, spherical VLPs (see Figure 2f).

[0089] <Example 2-6> To measure the degree to which the VLPs GII.4, GII.17, and GII.3 purified according to the present invention bind to HBGA, an HBGA binding assay was performed. As shown in Figure 2g, 100 µl each of five biotin-bound 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 placed in a streptavidin-coated 96-well plate (High Binding Capacity (HBC) Nutraavidin Plate (Thermo Scientific®)) at a concentration of 10 µg / ml, and the mixture was reacted at room temperature for 5 hours. After washing the plates with PBST, GII.17 VLP protein was diluted in PBS and added to each well at 100 μl, and reacted at 4°C for 18 hours. After washing the plates with PBST, rabbit NoV GII.17 Polyclonal IgG Ab was diluted 2000-fold in PBS and added to each well at 100 μl, and reacted 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 and added to each well at 100 μl, and treated at room temperature for 1 hour and 30 minutes. Subsequently, the reaction was allowed to develop color using TMB solution at room temperature for 5 minutes, then the color reaction was stopped with 0.5 M sulfuric acid solution, and the absorbance was measured at a wavelength of 450 nm. The binding between norovirus VLPs and five types of HBGAs was confirmed, and it was found 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 type1), respectively. The degree of binding is expressed in EC50, as shown in Figure 2h.

[0090] [Example 3] Confirmation of immunogenicity of GII.17 VLP formed using mRID <Example 3-1> To measure the ability to induce antibodies specific to the norovirus GII.17 antigen, 500 μl of virus-like particles (VLPs, consisting of purified antigen protein after the RID (EE domain + m3c)-NoV GII.17 VP1) of the present invention was cleaved with the fusion protein of the present invention) were administered via intramuscular injection to a group of 7-week-old SD (Sprague Dawley) rats (5 mice / group) at varying concentrations (Figure 3a). The prescribed dose volume (500 μL / head) was administered to both thighs of the animals using a syringe fitted with a 26-gauge needle, and 0.5 mg of norovirus GII.17 VLP antigen and aluminum hydroxide as an immunostimulant were mixed. The vaccine was administered in three doses at 2-week intervals, with approximately 1 mL of whole blood collected via the jugular vein on the scheduled date and injected into a vacuum tube containing a clot activator.

[0091] To measure the target protein-specific antibody titers in serum obtained from each animal experimental group, enzyme-linked immunosorbent assay (ELISA) was performed. Norovirus GII.17 VLP was added to 96-well plates (High Binding Capacity (HBC) Nutraavidin Plates (Thermo Scientific®)) at a concentration of 0.5 ug / ml, 100 ul per well, and coated at 4°C overnight. Then, 200 ul of 3% BSA solution was added, and the plates were blocked after reaction at 37°C for 2 hours. After washing the plates with PBST, the immunoserum was diluted (1:1600 dilution ratio), 100 ul per well, and reacted at 37°C for 1 hour and 30 minutes. After washing the plates with PBST, antibodies against goat-derived mouse IgG (southern Biotech 1030-05, Goat-anti mouse IgG HRP) were treated at 37°C for 1 hour to detect antibodies in serum bound to GII.17 VLP. Subsequently, the samples were color-developed at room temperature for 5 minutes using TMB (sigma T0440) solution, and the color reaction was stopped with 0.5 M sulfuric acid solution. The absorbance was then measured at a wavelength of 450 nm. After inoculation, GII.17 VLP-specific antibody titers in blood samples were confirmed at 8000-fold dilutions from test groups of 1 μg / head, 5 μg / head, 10 μg / head, and 20 μg / head. As shown in Figures 3ba to 3bc, it was confirmed that substantial GII.17 VLP antigen-specific antibodies were formed at all three concentrations of 5 ug, 10 ug, and 20 ug even with only secondary inoculation. In detail, in the 0.2 μg / head test group, antibody production was confirmed in all individuals from 14 days after the second dose.

[0092] <Example 3-2> To measure the degree to which immunoserum inhibits the binding between HBGA and GII.17 VLP antigen (VLP consisting of purified antigen protein after the RID (EE domain + m3c)-NoV GII.17 VP1 fusion protein of the present invention is cleaved, and then Nov VLP is shown in Figure 3c) and HBGA, an HBGA blocking assay was performed on immunoserum obtained on different dates after inoculation (Figure 3c). 100 µl of Blood type B(tri)-PAA-biotin(01-033);(Glycotech), which has high binding affinity to GII.17, was added to each well of a streptavidin-coated 96-well plate (High Binding Capacity (HBC) Neutraavidin Plate (Thermo Scientific®)) at a concentration of 10 µg / ml, and the mixture was reacted at room temperature for 5 hours. Primary, secondary, and tertiary immunosorbents, used to confirm the inhibitory effect of antigen-HBGA binding, were sequentially diluted 100-fold to 2-fold in PBS and reacted with GII.17 VLP protein diluted to the appropriate concentration at 37°C for 1 hour and 30 minutes. 100 µl of serum and GII.17 VLP reaction solution were added to the plate reacted with HBGA at 1 well, and the plates were reacted at 4°C for 18 hours. After washing the plates with PBST, rabbit NoV GII.17 Polyclonal IgG Ab was diluted 2000-fold in PBS and added to 100 µl per well, reacting 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 in PBS and added to 100 µl per well, treating at room temperature for 1 hour and 30 minutes. Subsequently, the sample was allowed to develop color using TMB solution at room temperature for 5 minutes, then the color reaction was stopped with 0.5 M sulfuric acid solution, and the absorbance was measured at a wavelength of 450 nm.

[0093] Figure 3d shows the results of an immunoserum HBGA binding inhibition test (using B tri synthetic carbohydrates), confirming HBGA inhibitory efficacy at 5 μg / head (low concentration), 10 μg / head (medium concentration), and 20 μg / head (high concentration), respectively, 14 days after primary, secondary, and tertiary vaccinations. As shown in Figure 3d, the HBGA binding inhibitory efficacy increased in a dose-dependent manner from the primary immunization serum. When the GII.17 VLP results were analyzed, an HBGA binding inhibitory efficacy of 1:200 or higher was confirmed at all doses after primary vaccination, a high HBGA binding inhibitory efficacy of 1:5,000 or higher was confirmed at all doses after secondary vaccination, and a high HBGA binding inhibitory efficacy of 1:6,000 or higher was confirmed at all doses after tertiary vaccination. The degree of inhibition of VLP antigen-HBGA binding was not large at all concentrations, but in secondary and tertiary immunization serum, the degree of inhibition of VLP antigen-HBGA binding was even higher at all concentrations compared to after primary vaccination. The difference in inhibitory efficacy between the low-concentration group and the medium- and high-concentration groups was large, and saturation was confirmed.

[0094] Referring to the results of Example 3, when the norovirus GII.17 antigen protein was expressed in a fusion protein form with the conventional RID, it was confirmed that it maintained activity as a fusion partner assisting in the folding of the target protein, exhibited excellent soluble expression levels, overcame protein misfolding, stabilized the protein structure, and improved folding and assembly capabilities. Through this, it was found that not only was the efficiency and homogeneity of VLP assembly by the final purified norovirus GII.17 VP1 protein excellent, but the antibodies induced through inoculation with the VLP also exhibited excellent performance in inhibiting binding to HBGA, which plays a key role in intracellular infection of norovirus.

[0095] [Example 4] Production of Norovirus GII.3 VP1 Virus-like Particles (VLPs) with Enhanced N-Terminal Expression and Soluble-Enhancing Partners for High Efficiency and Solubleness <Example 4-1> To create virus-like particles composed of the norovirus GII.3 VP1 protein, the VP1 gene (Norovirus Hu / GII.3 / CHDC2005 / 1975 / US, NCBI access number: HM072045.1) derived from the norovirus GII.3 protein was used. VP1, the antigen of norovirus, consists of 547 amino acids, and its protein molecular weight is approximately 59.7 kDa. Based on the aforementioned protein sequence, the synthesized gene sequence was produced after codon optimization in E. coli (SEQ ID NO: 10). (Gene synthesis was commissioned to Bionics Co., Ltd.)

[0096] In Figure 4a, recombinant fusion proteins were prepared by ligating an EE domain (MSAVKAA (SEQ ID NO: 2)) with increased expression and solubility at the N-terminus of a protein to an SE domain (SEQ ID NO: 3) 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 pattern was verified.

[0097] Referring to Figure 4a, it can be confirmed that the fusion protein (RID(EE domain+m3c)-NoV GII.3 VP1) bound to RID(EE domain+m3c)-(mutant RID peptide containing K23A, K30A, and K31A mutations) exhibits a significantly higher solubility(s) expression pattern than the fusion protein (RID-NoV GII.3 VP1) bound to RID without mutations.

[0098] Furthermore, using the ENLYFQG / S sequence, which is the TEV cleavage site, an optimized linker was used: a peptide with the amino acid sequence (GaSb)n (a≧1, b≧1, n≧1) was inserted between the TEV cleavage sequence (ENLYFQ) and the target protein (norovirus antigen protein) sequence. A schematic diagram of the fusion protein expression vector produced in this example is shown in Figure 1a.

[0099] The gene for the aforementioned sequence was constructed based on the pET-9a-EE domain(MSAVKAA)-SE domain(wt)-Nov GII.17 VP1, pET-9a-EE domain(MSAVKAA)-m3c-Nov GII.17 VP1 vector, which is a recombinant complex protein expression vector prepared in Example 1-1 using the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 1) expression vector. The T7 RNA polymerase is expressed by IPTG, and through this, the Lac operon and T7 promoter present on the DE3 genome are activated. The conventional N-terminus of the pET vector was inserted into the MCS (Multiple Cloning Site), and a RID sequence for increasing solubility and expression rate, and a ligation component sequence for enhancing 6xHIS and TEV cleavage efficiency were reinserted in the C direction using KpnI and BamHI cleavage enzymes. In this study, a linker was inserted between the NdeI and KpnI sequences of the cleavage enzyme sites within the MCS to enhance the cleavage efficiency of the RID, cleavage enzyme sites, and TEV enzyme. A DNA sequence designed to contain a protein in which the norovirus VP1 antigen protein is fused was inserted between the KpnI and BamHI cleavage enzymes. Each gene was then linked to the pET vector via T4 DNA ligase. DNA sequence analysis confirmed that the bacterial clones generated by these DNA linkage reactions had codon-optimized nucleotide sequences inserted.

[0100] The sequences of the recombinant complex expression vectors prepared in Example 4-1 are as follows:

[0101] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GII.3 VP1 [(Sequence ID 2)-(Sequence ID 3)-GT-6xHIS-TEV-GS-(Sequence ID 10)]: MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEINNEAMALEPVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGE FTVSPRNSPGEVLLNLELGPEINPYLAHLARMYNGYAGGFEVQVVLAGNAFTAGKVIFAAIPPNFPIDNLSAAQITMCPHVIVDVRQLEPINLPMPDVRNNFFHYNQGSDSRLRLIAMLYTPLRANNSGDDVFTVSCRVLTRPSPDFSFNFLVPPTVESKTK PFTLPILTISEMSNSRFPVPIDSLHTSPTENIVVQCQNGRVTLDGELMGTTQLLPSQICAFRGTLTRSTSRAGDQADTATPRLFNYYWHIQLDNLNGTPYDPAEDIPAPLGTPDFRGKVFGVASQRNPDSTTRAHEAKVDTTSGRFAPKLGSLEISTESSD FDSNQPTRFTPVGIGVDNEADFQQWSLPDYSGQFTHNMNLAPAVAPNFPGEQLLFFRSQLPSSGGRSNGILDCLVPQEWVQHFYQESAPAQTQVALVRYVNPDTGRVLFEAKLHKLGFMTIAKNGDSPITVPPNGYFRFESWVNPFYTLAPMGTGNGRRRIQ

[0102] The aforementioned manufactured composite was named "RID-NoV GII.3 VP1".

[0103] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GII.3 VP1 [(Sequence ID 2)-(Sequence ID 7)-GT-6xHIS-TEV-GS-(Sequence ID 10)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-KMASNDAAPSNDGAAGLVPEINNEAMALEPVAGAAIAAPLTGQQNIIDPWIMNNFVQAPGGEFTVSPRNSPGEVLLNLELGPEINPYLAHLARMYNGYAGGFEVQVVLAGNAFTAGKVIFA AIPPNFPIDNLSAAQITMCPHVIVDVRQLEPINLPMPDVRNNFFHYNQGSDSRLRLIAMLYTPLRANNSGDDVFTVSCRVLTRPSPDFSFNFLVPPTVESKTKPFTLPILTISEMSNSRFPVPIDSLHTSPTENIVVQCQNG RVTLDGELMGTTQLLPSQICAFRGTLTRSTSRAGDQADTATPRLFNYYWHIQLDNLNGTPYDPAEDIPAPLGTPDFRGKVFGVASQRNPDSTTRAHEAKVDTTSGRFAPKLGSLEISTESSDFDSNQPTRFTPVGIGVDNEA DFQQWSLPDYSGQFTHNMNLAPAVAPNFPGEQLLFFRSQLPSSGGRSNGILDCLVPQEWVQHFYQESAPAQTQVALVRYVNPDTGRVLFEAKLHKLGFMTIAKNGDSPITVPPNGYFRFESWVNPFYTLAPMGTGNGRRRIQ

[0104] The fabricated complex was named "RID(EE domain+m3c)-NoV GII.3 VP1".

[0105] <Example 4-2> The two expression vectors from Example 4-1 were used to transform 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 at 250 rpm for 5-7 hours. After that, 1 ml was transferred to 10 ml for a 10-fold dilution, and the cells were cultured again. After about 2-4 hours, when the O.D. 600 reached 0.5-0.7, 0.4 mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17-19 hours (maximum 21 hours). The culture medium was collected by centrifugation to obtain only the precipitated E. coli, which was then stored frozen at -80°C. 0.3 ml of lysis buffer solution (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 culture medium containing the corresponding E. coli harvest. After sonication, the total lysate (T) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) by centrifugation. Analysis by SDS-PAGE confirmed that the norovirus GII.3 VP1 protein (RID(EE domain+m3c)-NoV GII.3 VP1 fusion protein; right side of Figure 4a), with a mutant RID possessing an RNA binding site added to the N-terminus, exhibited highly efficient soluble expression, as shown in Figure 4a.

[0106] <Example 4-3> To obtain the RID(EE domain+m3c)-NoV GII.3 VP1 fusion protein, 500 ml of E. coli was cultured and expression induced using the same culture method as in Examples 1-2, which confirmed highly efficient soluble expression. The obtained cells 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 resuspended cells were pulverized using an ultrasonic cell disruptor. The lysed cells were centrifuged at 13500 rpm for 10 minutes at 4°C, and the supernatant was taken and subjected to Ni+ affinity chromatography using AKTA (GE Healthcare). First, a high-performance Ni Sepharose (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), and then the cell lysate supernatant was loaded onto the equilibrated column at a flow rate of 3-5 ml / min. Norovirus GII.3 VP1 protein was eluted and acquired by increasing injection in a linear concentration gradient in the imidazole concentration range of 10 mM to 300 mM using buffer B buffer [50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole] containing buffer A and 300 mM imidazole.

[0107] After confirming the separation pattern of the target protein in the eluted material via SDS-PAGE, the eluted material from that fraction was dialyzed overnight (12-16 hours) at 4°C in a dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol], and the bond between the N-terminal RID and the norovirus antigen protein VP1 was cleaved using a TEV cleavage enzyme.

[0108] To separate and purify the target protein from which the N-terminal fusion partner RID and other components have been removed, a high-performance Ni Sepharose (Cytiva) column was equilibrated with the same solution A buffer as the dialysis buffer [50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% 2-mercaptoethanol], and then loaded onto the equilibrated column at a flow rate of 3-5 ml / min. The NoV GII.-3 target protein from which the N-terminal fusion partner RID and other components have been removed was recovered in a form that eluted without being adsorbed onto the column.

[0109] 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 A (50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol), and then loaded at a flow rate of 1 ml / min. The target protein was eluted by NaCl injection with a linear concentration gradient of 10 mM to 1 M through buffers AA and BB (50 mM Tris-HCl (pH 8.5), 1 M NaCl, 5% glycerol, 0.1% β-mercaptoethanol), and obtained in 1 ml fractions. The purification characteristics and purity are shown in Figures 4b, 4c, and 4d. The concentration of the final purified protein was quantified using BSA (Amresco, Solon, OH, USA). As can be seen from Figures 4b to 4d, the RID protein bound within the fusion protein was separated, indicating that the target protein was successfully purified.

[0110] <Example 4-4> The purified norovirus VP1 protein was stored at 4°C in a final assembly buffer [100 mM potassium phosphate, pH 5.8, 150 mM NaCl, 10% glycerol] containing 10% glycerol, with the buffer being changed overnight (12-16 hours). The concentration of the final purified protein was quantified using BSA (Amresco, Solon, OH, USA).

[0111] <Examples 4-5> To confirm the overall diameter distribution of virus-like particles (VLPs) self-assembled by the target protein purified after RID cleavage using the fusion protein (Norovirus VP1 fusion protein containing RID mutant protein (RID(EE domain+m3c)-NoVGII.3 VP1 fusion protein)) prepared in Example 4-4, analysis was performed via Dynamic Light Scattering (DLS). The analysis of the complex was performed using DLS (Particular Systems, Zetasizer Nano Family) with 1 mL of sample placed in a cuvette at a temperature of 16°C. Dynamic light scattering analysis of cells was confirmed using the total intensity and mass of each region in the image. As shown in Figure 4e, it was confirmed that the particles formed had a size distribution of 30-40 nm in diameter at the hydrodynamic radius of the DLS.

[0112] <Example 4-6> The external shape of virus-like particles (VLPs) assembled with the target protein purified using the fusion protein (norovirus VP1 fusion protein containing the RID mutant protein (RID(EE domain+m3c)-NoV GII.3 VP1 fusion protein)) according to Example 4-4 was observed using an electron microscope. 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 (TEM (120kV); Talos L120C, FEI, Czech). As shown in Figure 4f, it was confirmed that the norovirus VP1 protein assembled after purification formed VLPs. The diameter of the observed virus-like particles (VLPs) was found to be between 30 and 40 nm, which is the diameter of norovirus particles, confirming the formation of highly homogeneous spherical VLPs.

[0113] The results of Example 4, similar to those of Example 2, confirm that when the mutant RID according to the present invention is fused with the norovirus antigen protein, it leads to exceptionally high-efficiency target protein expression, proper folding induction, and improved VLP assembly ability in E. coli.

[0114] [Example 5] Confirmation of immunogenicity of GII.3 VLP formed using mRID <Example 5-1> To measure the ability to induce antibodies specific to the norovirus GII.3 antigen, 500 μl of virus-like particles (VLPs, consisting of purified antigen protein after RID cleavage with the fusion protein of the present invention (RID(EE domain+m3c)-NoV GII.3 VP1)) produced according to the present invention were administered intramuscularly to a group of 7-week-old SD (Sprague Dawley) rats (5 mice / group) at various concentrations (Figure 5a). The prescribed dose volume (500 μL / head) was administered to both thighs of the animals using a syringe fitted with a 26-gauge needle, and 0.5 mg of norovirus GII.3 VLP antigen and aluminum hydroxide as an immunostimulant were mixed. The vaccine was administered in a total of three doses at 2-week intervals to induce immunization, and approximately 1 mL of whole blood was collected via the jugular vein at the scheduled time and injected into a vacuum tube containing a clot activator.

[0115] To measure the target protein-specific antibody titers in serum obtained from each animal experimental group, enzyme-linked immunosorbent assay (ELISA) was performed. Norovirus GII.3 VLP was added to 96-well plates (High Binding Capacity (HBC) Nutraavidin Plates (Thermo Scientific®)) at a concentration of 0.5 ug / ml, 100 ul per well, and coated at 4°C overnight. Then, 200 ul of 3% BSA solution was added, and the plates were blocked after reaction at 37°C for 2 hours. After washing the plates with PBST, the immunoserum was diluted (1:1600 dilution ratio), 100 ul per well, and reacted at 37°C for 1 hour and 30 minutes. After washing the plates with PBST, antibodies against goat-derived mouse IgG (southern Biotech 1030-05, Goat-anti mouse IgG HRP) were treated at 37°C for 1 hour to detect antibodies in serum bound to GII.3 VLP. Subsequently, the samples were color-developed at room temperature for 5 minutes using TMB (sigma T0440) solution, and the color reaction was stopped with 0.5 M sulfuric acid solution. The absorbance was then measured at a wavelength of 450 nm. After inoculation, GII.3 VLP-specific antibody titers in blood samples were examined at 8000-fold dilutions from test groups of 0.2 μg / head, 1 μg / head, 5 μg / head, 10 μg / head, and 20 μg / head. As shown in Figure 5ba, specific IgG antibody titers were confirmed in groups administered 1 μg or more after primary inoculation, and high specific IgG antibody titers were confirmed in all dose groups after secondary inoculation (Figures 5bb, 5bc).

[0116] <Example 5-2> To measure the degree to which immunoserum inhibits the binding between GII.3 VLP antigen (VLP consisting of the antigen protein purified after the RID (EE domain + m3c)-NoV GII.3 VP1 fusion protein of the present invention is cleaved with the RID, Nov GII.3 VLP in Figure 5c) and HBGA, an HBGA blocking assay was performed on immunoserum obtained on different dates after inoculation (Figure 5c). Lea-PAA-biotin (01-035); (Glycotech), which has a high binding affinity to type GII.3, was added to each well of a streptavidin-coated 96-well plate (High Binding Capacity (HBC) Neutraavidin Plate (Thermo Scientific®)) at a concentration of 10 ug / ml, with 100 ul added per well, and the plate was reacted at room temperature for 5 hours. Primary, secondary, and tertiary immunosorbents, each used to confirm the inhibitory effect of VLP antigen-HBGA binding, were sequentially diluted 100-fold to 2-fold in PBS and reacted with GII.3 VLP protein at appropriate concentrations at 37°C for 1 hour and 30 minutes. 100 µl of serum and GII.3 VLP reaction solution were added per well to a plate reacted with HBGA, and the reaction was incubated at 4°C for 18 hours. After washing the plate with PBST, rabbit NoV GII.3 Polyclonal Ig G Ab was diluted 2000-fold in PBS and added to 100 µl per well, reacting at room temperature for 1 hour and 30 minutes. After washing with PBST, goat-anti-rabbit IgG HRP was diluted 30000-fold in PBS and added to 100 µl per well, treating at room temperature for 1 hour and 30 minutes. Subsequently, the reaction was allowed to develop color using TMB solution at room temperature for 5 minutes, then the color reaction was stopped with 0.5 M sulfuric acid solution, and the absorbance was measured at a wavelength of 450 nm.

[0117] Figure 5c shows the results of the immunoserum HBGA binding inhibition test (using Le a synthetic carbohydrate), confirming the HBGA inhibitory efficacy at 5 μg / head (low concentration), 10 μg / head (medium concentration), and 20 μg / head (high concentration) 14 days after primary, secondary, and tertiary vaccination, respectively. GII.3 Analysis results showed that after primary vaccination, HBGA binding inhibition efficacy of 1:200 or higher was confirmed at all doses. After secondary vaccination, significantly increased HBGA binding inhibition efficacy of 1:1,500 or higher was confirmed at all doses compared to the primary dose. After tertiary vaccination, high HBGA binding inhibition efficacy of 1:2,500 or higher was confirmed at all doses. Similar HBGA binding inhibitory effects were observed in all dose groups after primary vaccination. After secondary vaccination, exceptionally high efficacy was induced compared to the primary vaccination, and even higher HBGA binding inhibition efficacy was induced in the 10 μg and 20 μg dose groups compared to the 5 μg dose group. After the third dose, an even higher HBGA binding inhibitory effect was induced, and regardless of the administered dose, it generally showed signs of saturation, confirming that the antigen exhibited high immunogenicity.

[0118] In the results of Example 5, similar to the results of Example 3, it was confirmed that when the norovirus GII.3 antigen protein according to the present invention is expressed in a fusion protein form with an existing RID, it maintains activity as a fusion partner that assists in the folding of the target protein, exhibits excellent soluble expression levels, overcomes protein misfolding, stabilizes the protein structure, and improves folding and assembly capabilities. Through this, it was found that not only is the efficiency and homogeneity of VLP assembly by the final purified norovirus GII.3 VP1 protein excellent, but the antibodies induced through inoculation with the VLP exhibit excellent performance in inhibiting binding to HBGA, which plays a key role in intracellular infection of norovirus.

[0119] [Example 6] Production of VP1 virus-like particles (VLPs) based on the genotype of norovirus antigen target protein purified with fusion protein To create virus-like particles consisting of a target protein (norovirus antigen protein) purified with a norovirus VP1 protein fusion protein to which a RID sequence for highly efficient soluble expression is attached at the N-terminus, VP1 genes derived from the proteins of each norovirus genotype were used: 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).

[0120] <Example 6-1> In Figure 6a, the VP1 gene derived from the norovirus genotype GI.1 (Norovirus Hu / GI.1 / 8McIII / 1973 / USA, NCBI access number: NC_001959.2) protein was used. VP1, the norovirus antigen, consists of approximately 500 amino acids, and its protein molecular weight is approximately 55-60 kDa. Based on the aforementioned protein sequence, the synthesized gene sequence was produced in E. coli after codon optimization (SEQ ID NO: 12). (Gene synthesis was commissioned to Bionics Co., Ltd.)

[0121] The gene of the aforementioned sequence was prepared using the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 1) expression vector as the basis for the recombinant complex protein expression vectors pET-9a-EE domain(MSAVKAA)-SE domain(WT)-Nov GII.17 VP1 and pET-9a-EE domain(MSAVKAA)-m3c-Nov GII.17 VP1 vectors prepared in Example 1-1. T7 RNA polymerase is expressed by IPTG, and through this, the Lac operon and T7 promoter present on the DE3 genome are activated. The RID sequence for increasing solubility and expression rate at the conventional N-terminus, and the 6xHIS and ligating component sequences for enhancing TEV cleavage efficiency are excised in the C direction using KpnI and BamHI cleavage enzymes, and then subcloned and reinserted. In this study, a linker was inserted between the NdeI and KpnI sequences of the cleavage enzyme sites within the MCS to enhance the cleavage efficiency of the RID, cleavage enzyme sites, and TEV enzyme. A DNA sequence designed to contain a protein in which the norovirus VP1 antigen protein is fused was inserted between the KpnI and BamHI cleavage enzymes. Each gene was then linked to the pET vector via T4 DNA ligase. DNA sequence analysis confirmed that the bacterial clones generated by these DNA linkage reactions had codon-optimized nucleotide sequences inserted.

[0122] Recombinant proteins were prepared by fusing a RID EE domain peptide (MSAVKAA (SEQ ID NO: 2)), which has the effect of increasing expression level and solubility at the N-terminus of the protein, with a RID SE domain peptide (SEQ ID NO: 3) or a mutant RID peptide (SEQ ID NO: 7) that is more than 50% similar to SEQ ID NO: 3 and contains K23A, K30A, and K31A mutations, and their soluble expression patterns were verified.

[0123] Furthermore, using the ENLYFQG / S sequence, which is the TEV cleavage site, an optimized linker is used: a peptide with the amino acid sequence (GaSb)n (a≧1, b≧1, n≧1) is placed between the TEV cleavage sequence and the target protein (norovirus antigen protein) sequence.

[0124] The sequences of 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 of the aforementioned sequence are the following two:

[0125] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GI.1 VP1 [(Sequence ID 2)-(Sequence ID 3)-GT-6xHIS-TEV-GS-(Sequence ID 12)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-MMMASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWII NNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGNAFTAGKIIVSCIPPGFGSHNLTIAQATLPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFN FLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMGISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGV LSWISPPSHPSGSQVDLWKIPNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR

[0126] The aforementioned manufactured composite was named "RID-NoV GI.1 VP1".

[0127] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GI.1 VP1 [(Sequence ID 2)-(Sequence ID 7)-GT-6xHIS-TEV-GS-(Sequence ID 12)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-MMMASKDATSSVDGASGAGQLVPEVNASDPLAMDPVAGSSTAVATAGQVNPIDPWIINNFVQAPQGEFTISPNNTPGDVLFDLSLGPHLNPFLLHLSQMYNGWVGNMRVRIMLAGN AFTAGKIIVSCIPPGFGSHNLTIAQATLFPHVIADVRTLDPIEVPLEDVRNVLFHNNDRNQQTMRLVCMLYTPLRTGGGTGDSFVVAGRVMTCPSPDFNFLFLVPPTVEQKTRPFTLPNLPLSSLSNSRAPLPISSMG ISPDNVQSVQFQNGRCTLDGRLVGTTPVSLSHVAKIRGTSNGTVINLTELDGTPFHPFEGPAPIGFPDLGGCDWHINMTQFGHSSQTQYDVDTTPDTFVPHLGSIQANGIGSGNYVGVLSWISPPSHPSGSQVDLWKI PNYGSSITEATHLAPSVYPPGFGEVLVFFMSKMPGPGAYNLPCLLPQEYISHLASEQAPTVGEAALLHYVDPDTGRNLGEFKAYPDGFLTCVPNGASSGPQQLPINGVFVFVSWVSRFYQLKPVGTASSARGRLGLRR

[0128] The fabricated complex was named "RID(EE domain+m3c)-NoV GI.1 VP1".

[0129] <Example 6-2> The two expression vectors from Example 5-1 were used to transform 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 at 250 rpm for 5-7 hours. After that, 1 ml was transferred to 10 ml for a 10-fold dilution, and the cells were cultured again. After about 2-4 hours, when the O.D. 600 reached 0.5-0.7, 0.4 mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17-19 hours (maximum 21 hours). The culture medium was collected by centrifugation to obtain only the precipitated E. coli, which was then stored frozen at -80°C. 0.3 ml of lysis buffer solution (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 culture medium containing the corresponding E. coli harvest. After ultrasonic grinding, the total lysate (T) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) by centrifugation. Analysis by SDS-PAGE confirmed that the norovirus GI.1 VP1 protein with a mutant RID added to the N-terminus exhibited highly efficient soluble expression, as shown in Figure 5a.

[0130] <Example 6-3> In Figure 6b, the VP1 gene derived from the norovirus genotype GI.3 (Norwalk-like virus NLV / Honolulu / 219 / 1992 / US, NCBI access number: AF414403) protein was used. VP1, the norovirus antigen, consists of approximately 500 amino acids, and its protein molecular weight is approximately 55-60 kDa. Based on the aforementioned protein sequence, the synthesized gene sequence was produced in E. coli after codon optimization (SEQ ID NO: 13). (Gene synthesis was commissioned to Bionics Co., Ltd.)

[0131] The gene of the aforementioned sequence was constructed using the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 1) expression vector as the basis for the recombinant complex protein expression vectors pET-9a-MSAVKAA-RID-Nov GII.17 VP1 and pET-9a-MSAVKAA--Nov GII.17 vector, which were prepared in Example 1. T7 RNA polymerase is expressed by IPTG, and through this, the RID sequence for increasing solubility and expression rate, and the 6xHIS and TEV cleavage efficiency linking component sequences, which are inserted into the MCS (Multiple Cloning Site) of the pET vector, which is activated by the Lac operon and T7 promoter present on the DE3 genome, are excised in the C direction using KpnI and BamHI cleavage enzymes in the conventional N-terminus. These sequences were then subcloned and reinserted. In this study, a linker was inserted between the NdeI and KpnI sequences of the cleavage enzyme sites within the MCS to enhance the cleavage efficiency of the RID, cleavage enzyme sites, and TEV enzyme. A DNA sequence designed to contain a protein in which the norovirus VP1 antigen protein is fused was inserted between the KpnI and BamHI cleavage enzymes. Each gene was then linked to the pET vector via T4 DNA ligase. DNA sequence analysis confirmed that the bacterial clones generated by these DNA linkage reactions had codon-optimized nucleotide sequences inserted.

[0132] Recombinant proteins were prepared by fusing MSAVKAA (SEQ ID NO: 2), which has the effect of increasing expression level and solubility at the N-terminus of the protein, with a RID peptide (SEQ ID NO: 3) or a mutant SE domain peptide (SEQ ID NO: 7) that is more than 50% similar to SEQ ID NO: 3 and contains mutations in K23A, K30A, and K31A, and their soluble expression patterns were verified.

[0133] Furthermore, using the ENLYFQG / S sequence, which is the TEV cleavage site, an optimized linker is used: a peptide with the amino acid sequence (GaSb)n (a≧1, b≧1, n≧1) is placed between the TEV cleavage sequence and the target protein sequence (norovirus antigen protein).

[0134] The sequences of the recombinant complex expression vectors confirmed in Example 5-3 using the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 3) expression vector containing the target protein of the aforementioned sequence are the following two:

[0135] EE domain(MSAVKAA)-SE domain-GT-6xHIS-TEV-GS-NoV GI.3 VP1 [(Sequence ID 2)-(Sequence ID 3)-GT-6xHIS-TEV-GS-(Sequence ID 13)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-MMMASKDAPPNMDGTSGAGQLVPEANTAEPISMEPVAGAATAAATAGQVNMIDPWIMNNYV QAPQGEFTISPNNTPGDILFDLQLGPHLNPFLSHLAQMYNGWVGNMKVKVLLAGNAFTAGKIIISCIPPGFAAQNISIAQATMFPHVIADVRVLEPIEVPLEDVRNVLFHNNDNTPTMRLVCMLYTPLRASGSSSGTDPFVIAGRVLTCPSPDFSLLFLVP PNVEQKTKPFSVPNLPLNTLSNSRVPSLIKSMMVSRDHGQMVQFQNGRVALDGQLQGTTPTSASQLCKIRGSVFHANGGNGYNPTELDGSPYHAFESPAPIGFPDLGECDWHMEASPTTQFDTGDVIKQINVKQESAFAPHLGTIQADGLSDVSVNTNMIA KLGWVSPVSDGHRGNVDPWVIPRYGSTLTEAAQLAPPIYPPGFGEAIVFFMSDFPIAHGTNGLSVPCTIPQEFVTHFVNEQAPTRGEAALLHYLDPDTHRNLGEFKLYPEGFMTCVPNSSGTGPQTLPINGVFVFVSWVSRFYQLKPVGTAGPARRLGIRRS

[0136] The aforementioned manufactured composite was named "RID-NoV GI.3 VP1".

[0137] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GI.3 VP1 [(Sequence ID 2)-(Sequence ID 7)-GT-6xHIS-TEV-GS-(Sequence ID 13)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-MMMASKDAPPNMDGTSGAGQLVPEANTAEPISMEPVAGAATAAATAGQVNMIDPWIMNNYVQAPQGEFTISPNNTPGDILFDLQLGPHLNPFLSHLAQMYNGWVGNMKVKVLLAGNAFTA GKIIISCIPPGFAAQNISIAQATMFPHVIADVRVLEPIEVPLEDVRNVLFHNNDNTPTMRLVCMLYTPLRASGSSSGTDPPFVIAGRVLTCPSPDFSLLFLVPPNVEQKTKPFSVPNLPLNTLSNSRVPSLIKSMMVSRDHGQ MVQFQNGRVALDGQLQGTTPTSASQLCKIRGSVFHANGGNGYNPTELDGSPYHAFESPAPIGFPDLGECDWHMEASPTTQFDTGDVIKQINVKQESAFAPHLGTIQADGLSDVSVNTNMIAKLGWVSPVSDGHRGNVDPWV IPRYGSTLTEAAQLAPPIYPPGFGEAIVFFMSDFPIAHGTNGLSVPCTIPQEFVTHFVNEQAPTRGEAALLHYLDPDTHRNLGEFKLYPEGFMTCVPNSSGTGPQTLPINGVFVFVSWVSRFYQLKPVGTAGPARRLGIRRS

[0138] The aforementioned fabricated complex was named "RID(EE domain+m3c)-NoV GI.3 VP1".

[0139] <Example 6-4> The two expression vectors from Example 6-3 were used to transform 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 at 250 rpm for 5-7 hours. After that, 1 ml was transferred to 10 ml for a 10-fold dilution, and the cells were cultured again. After about 2-4 hours, when the O.D. 600 reached 0.5-0.7, 0.4 mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17-19 hours (maximum 21 hours). The culture medium was collected by centrifugation to obtain only the precipitated E. coli, which was then stored frozen at -80°C. 0.3 ml of lysis buffer solution (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 culture medium containing the corresponding E. coli harvest. After ultrasonic grinding, the total lysate (T) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) by centrifugation. Analysis by SDS-PAGE confirmed that the norovirus GI.3 VP1 protein with a mutant RID added to the N-terminus exhibited highly efficient soluble expression, as shown in Figure 5b.

[0140] <Example 6-5> In Figure 6c, the VP1 gene derived from the Norovirus genotype GII.2 protein (Norovirus GII.2 strain Env / CHN / 2016 / GII.P16-GII.2 / BJSMQ, NCBI access number:NC_039476) was used. VP1, the antigen of norovirus, consists of approximately 500 amino acids, and its protein molecular weight is approximately 55-60 kDa. Based on the aforementioned protein sequence, the synthesized gene sequence was produced in E. coli after codon optimization (SEQ ID NO: 11). (Gene synthesis was commissioned to Bionics Co., Ltd.)

[0141] The gene of the aforementioned sequence was constructed using the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 1) expression vector as the basis for the recombinant complex protein expression vector pET-9a-MSAVKAA-RID-Nov GII.17 VP1,pET-9a-MSAVKAA-m3c-Nov GII.17 vector, which was constructed in Example 1. T7 RNA polymerase is expressed by IPTG, and through this, the Lac operon and T7 promoter present on the DE3 genome are activated. The RID sequence for increasing solubility and expression rate, and the 6xHIS and ligating component sequence for enhancing TEV cleavage efficiency are inserted into the MCS (Multiple Cloning Site) of the pET vector. These sequences are then excised in the C direction using KpnI and BamHI cleavage enzymes, subcloned, and reinserted. In this study, a linker was inserted between the NdeI and KpnI sequences of the cleavage enzyme sites within the MCS to enhance the cleavage efficiency of the RID, cleavage enzyme sites, and TEV enzyme. A DNA sequence designed to contain a protein in which the norovirus VP1 antigen protein is fused was inserted between the KpnI and BamHI cleavage enzymes. Each gene was then linked to the pET vector via T4 DNA ligase. DNA sequence analysis confirmed that the bacterial clones generated by these DNA linkage reactions had codon-optimized nucleotide sequences inserted.

[0142] Recombinant proteins were prepared by fusing an EE domain peptide (MSAVKAA (SEQ ID NO: 2)) with increased expression and solubility at the N-terminus of a target protein (norovirus antigen protein) with an SE domain peptide (SEQ ID NO: 3) 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 their soluble expression patterns were verified.

[0143] Furthermore, using the ENLYFQG / S sequence, which is the TEV cleavage site, an optimized linker is used: a peptide with the amino acid sequence (GaSb)n (a≧1, b≧1, n≧1) is placed between the TEV cleavage sequence and the target protein sequence (norovirus antigen protein).

[0144] The sequences of the recombinant complex expression vectors confirmed in Example 6-5 using the pET-9a(Novagene, 69431-3CN)(SEQ ID NO: 1) expression vector containing the target protein of the aforementioned sequence are the following two:

[0145] MSAVKAA-RID-GT-6xHIS-TEV-GS-NoV GII.2 VP1 [(Sequence ID 2)-(Sequence ID 3)-GT-6xHIS-TEV-GS-(Sequence ID 11)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV-GT-HHHHHH-ENLYFQ-GS-MKMASNDAAPSTDGAAGLVPESNNEVMALEPVAGAALAAPVTGQTNIIDPWIRANFVQAP NGEFTVSPRNAPGEVLLNLELGPELNPYLAHLARMYNGYAGGMEVQVMLAGNAFTAGKLVFAAVPPHFPVENLSPQQITMFPHVIIDVRTLEPVLLPLPDVRNNFFHYNQKDDPKMRIVAMLYTPLRSNGSGDDVFTVSCRVLTRPSPDFDFTYLVPPTVE SKTKPFTLPILTLGELSNSRFPVSIDQMYTSPNEIISVQCQNGRCTLDGELQGTTQLQVSGICAFKGEVTAHLHDNDHLYNVTITNLNGSPFDPSEDIPAPLGVPDFQGRVFGIISQRDKHNSPGHNEPANRGHDAVVPTYTAQYTPKLGQIQIGTWQTD DLTVNQPVKFTPVGLNDTEHFNQWVVPRYAGALNLNTNLAPSVAPVFPGERLLFFRSYIPLKGGYGNPAIDCLLPQEWVQHFYQEAAPSMSEVALVRYINPDTGRALFEAKLHRAGFMTVSSNTSAPVVVPANGYFRFDSWVNQFYSLAPMGTGNGRRRVQ

[0146] The aforementioned manufactured composite was named "RID-NoV GII.2 VP1".

[0147] MSAVKAA-m3c-GT-6xHIS-TEV-GS-NoV GII.2 VP1 [(Sequence ID 2)-(Sequence ID 7)-GT-6xHIS-TEV-GS-(Sequence ID 11)] :MSAVKAA-AAVQAAEVKVDGSEPKLSKNELARRLKAEAAVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV -GT-HHHHHH-ENLYFQ-GS-MKMASNDAAPSTDGAAGLVPESNNEVMALEPVAGAALAAPVTGQTNIIDPWIRANFVQAPNGEFTVSPRNAPGEVLLNLELGPELNPYLAHLARMYNGYAGGMEVQVMLAGNAFTAGKL VFAAVPPHFPVENLSPQQITMFPHVIIDVRTLEPVLLPLPDVRNNFFHYNQKDDPKMRIVAMLYTPLRSNGSGDDVFTVSCRVLTRPSPDFDFTYLVPPTVESKTKPFTLPILTLGELSNSRFPVSIDQMYTSPNEIISVQ CQNGRCTLDGELQGTTQLQVSGICAFKGEVTAHLHDNDHLYNVTITNLNGSPFDPSEDIPAPLGVPDFQGRVFGIISQRDKHNSPGHNEPANRGHDAVVPTYTAQYTPKLGQIQIGTWQTDDLTVNQPVKFTPVGLNDTEH FNQWVVPRYAGALNLNTNLAPSVAPVFPGERLLFFRSYIPLKGGYGNPAIDCLLPQEWVQHFYQEAAPSMSEVALVRYINPDTGRALFEAKLHRAGFMTVSSNTSAPVVVPANGYFRFDSWVNQFYSLAPMGTGNGRRRVQ

[0148] The fabricated complex was named "RID(EE domain+m3c)-NoV GII.2 VP1".

[0149] <Example 6-6> The two expression vectors from Example 6-5 were used to transform 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 at 250 rpm for 5-7 hours. After that, 1 ml was transferred to 10 ml for a 10-fold dilution, and the cells were cultured again. After about 2-4 hours, when the O.D. 600 reached 0.5-0.7, 0.4 mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added, and the cells were cultured at 16°C for 17-19 hours (maximum 21 hours). The culture medium was collected by centrifugation to obtain only the precipitated E. coli, which was then stored frozen at -80°C. 3 ml of culture medium was mixed with 0.3 ml of lysis buffer solution (50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 5% glycerol, 0.1% β-mercaptoethanol, and 10 mM imidazole). After ultrasonic grinding, the total lysate (T) was separated into a precipitate (P) and a supernatant (S) by centrifugation. Analysis by SDS-PAGE confirmed that the norovirus GII.2 VP1 protein (fusion protein) with a mutant RID added to the N-terminus exhibited highly efficient soluble expression, as shown in Figure 5c.

[0150] <Examples 6 and 7> The VP1 proteins of norovirus GI.1, GI.3, and GII.2 obtained in Example 6 were purified using the same purification process as in the above example. The purified norovirus VP1 proteins were then transferred to assembly buffer containing 10% glycerol and stored overnight (12-16 hours) at 4°C. The assembled virus-like particles (VLPs) were then analyzed by dynamic light scattering (DLS) to confirm the overall diameter distribution. The analysis of the complex was performed using DLS (Particular Systems, Zetasizer Nano Family) with 1 mL of sample placed in a cuvette at a temperature of 16°C. Dynamic light scattering analysis of cells was confirmed using the total intensity and mass of each region in the image. The external shape of the virus-like particles (VLPs) was also observed using an electron microscope. 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 imaged using a transmission electron microscope (TEM, 120kV; Talos L120C, FEI, Czech). As shown in Figures 6da to 6dc, it was confirmed that the norovirus VP1 protein assembled after purification had a particle size distribution of 30-40 nm in diameter at the hydrodynamic radius of the DLS, and formed VLPs. The diameter of the VLPs confirmed by TEM was found to be between 30-40 nm, which is the diameter of norovirus particles, confirming the formation of highly homogeneous spherical VLPs.

[0151] When the mutant RID according to the present invention fuses with the norovirus antigen protein, it can be confirmed that this is essential for exceptionally efficient expression of the target protein in E. coli, proper folding induction, and improved VLP assembly ability.

[0152] Although embodiments of the present invention have been described above, those with ordinary skill in the art to which the present invention pertains will understand that the present invention may be carried out in other specific forms without altering its technical idea or essential features. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

[0153] Sequence List Sequence ID 1 TIFF2026514122000003.tif131147 TIFF2026514122000004.tif234147 TIFF2026514122000005.tif42143

[0154] Sequence ID 2 TIFF2026514122000006.tif12143

[0155] Sequence ID 3 TIFF2026514122000007.tif18143

[0156] Sequence ID 4 TIFF2026514122000008.tif18143

[0157] Sequence ID 5 TIFF2026514122000009.tif18143

[0158] Sequence ID 6 TIFF2026514122000010.tif18143

[0159] Sequence ID 7 TIFF2026514122000011.tif18143

[0160] Sequence ID 8 TIFF2026514122000012.tif18147

[0161] Sequence ID 9 TIFF2026514122000013.tif81147

[0162] Sequence ID 10 TIFF2026514122000014.tif80147

[0163] Sequence ID 11 TIFF2026514122000015.tif80147

[0164] Sequence ID 12 TIFF2026514122000016.tif80147

[0165] Sequence ID 13 TIFF2026514122000017.tif79143

Claims

1. Norovirus antigen protein; and A recombinant expression vector for norovirus vaccine production containing a polynucleotide encoding a mutant protein in which three or four amino acid residues in the RNA interacting domain (RID) sequence isolated from mammalian lysyl tRNA synthetase (mammalian LysRS) are replaced with other amino acid residues.

2. The recombinant expression vector for norovirus vaccine production according to claim 1, wherein the RID consists of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:

3.

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

3.

4. The recombinant expression vector for norovirus vaccine production according to claim 1, wherein the mutant protein of RID contains one of the amino acid sequences of SEQ ID NOs. 5 to SEQ ID NOs.

8.

5. The recombinant expression vector for norovirus vaccine production according to claim 1, wherein the norovirus antigen protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 13.

6. A host cell transformed with an expression vector according to any one of claims 1 to 5.

7. The host cell according to claim 6, wherein the host cell is Escherichia coli (E. coli).

8. Norovirus antigen protein; and A fusion protein for norovirus vaccines containing a mutant protein in which three or four amino acid residues in the RNA interacting domain (RID) sequence isolated from mammalian lysyl tRNA synthetase (mammalian LysRS) are replaced with other amino acid residues.

9. The aforementioned RID consists of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:

3. The fusion protein according to claim 8, wherein the substituted amino acid residue includes at least one of the amino acid residues at positions 19, 23, 24, 30, 31, 38, and 40 in the amino acid sequence of Sequence ID No.

3.

10. The fusion protein according to claim 8, wherein the mutant protein of RID consists of at least one amino acid sequence selected from the group consisting of SEQ ID NOs. 5 to SEQ ID NOs.

8.

11. The fusion protein according to claim 8, wherein the norovirus antigen protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 13.

12. A virus-like particle formed by the self-assembly of an antigen protein cleaved by the fusion protein described in claim 8.

13. (a) A step of producing a recombinant expression vector for vaccine production containing 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 lysyl tRNA synthetase (mammalian LysRS) are replaced with other amino acid residues; (b) the step of introducing the expression vector into host cells to produce transformants; and (c) A method for producing norovirus-like particles, comprising the step of culturing the transformant, inducing the expression of a recombinant fusion protein, and then obtaining the transformed substance.

14. The aforementioned fusion protein contains a TEV (tobacco etch virus) protein between the norovirus antigen protein and the RID protein. The method for producing norovirus-like particles according to claim 13, further comprising the step of (c) after step (d) cleaving the RID protein and norovirus antigen protein of the produced fusion protein using a TEV cleavage enzyme.

15. A method for producing norovirus-like particles according to claim 14, comprising the step of (e) purifying the cleaved antigen protein after step (d).

16. A method for producing norovirus-like particles according to claim 15, further comprising the step after step (e) above, in which the purified protein is self-assembled to form virus-like particles.

17. The aforementioned RID consists of the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO:

3. The method for producing norovirus-like particles according to claim 13, wherein the substituted amino acid residue includes at least one of the amino acid residues at positions 19, 23, 24, 30, 31, 38, and 40 in the amino acid sequence of Sequence ID No.

3.

18. The method for producing norovirus-like particles according to claim 13, wherein the mutant protein of RID contains one of the amino acid sequences from SEQ ID NOs. 5 to SEQ ID NOs.

8.

19. The method for producing norovirus-like particles according to claim 13, wherein the norovirus antigen protein comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 13.

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