KR20210027606A - Pentamer-based recombinant protein vaccine platform and expressing system there of - Google Patents

Abstract

The present invention relates to a recombinant expression vector for producing a pentamer-based recombinant protein vaccine, a host cell into which the expression vector is introduced, and a method for producing a pentamer-based recombinant protein vaccine using the vector and the host cell. According to the present invention, by greatly improving the expression level and water solubility of the recombinant protein in prokaryotic cells, an antigen protein can be produced in high yield, and a structurally sophisticated pentameric antigen protein can also be produced. In addition, the immunogenicity of a virus vaccine comprising a pentameric fusion protein of the present invention can be greatly improved.

Description

Pentamer-based recombinant protein vaccine platform and expressing system there of}

The present invention relates to a gene recombinant protein expression vector system based on self-assembly of a pentamer having high water solubility and immunogenicity, and a method of using the same.

Among the vaccine development methods, the method of producing virus-derived antigen proteins and using them as vaccines uses eukaryotic cells such as mammalian cells, plant cells, insect cells, yeast, and prokaryotic cells such as E. coli through gene recombination. . Each different production system has its own strengths and weaknesses. In particular, when higher cells such as mammalian cells or plant cells are used, it is possible to express sophisticated antigen proteins in water-soluble manner, but there is a disadvantage that requires a lot of cost and time. On the other hand, in the case of the E. coli system, a very large amount of protein can be produced quickly and inexpensively, but most of the proteins lose their water solubility and are produced by insoluble precipitation. In particular, eukaryotic proteins or viral proteins that cause infection therewith have limitations in inducing a folding structure and self-assembly of proteins in a prokaryotic system having a relatively simple structure.

Toxin proteins belonging to the heat-labile enterotoxin (LT) family are generally composed of an AB5 type structure, where A is the toxin that is toxic to animals and B5 is the carrier B that carries the toxin A to the human body. It means a pentameric structure (Faruque SM: Nair GB, eds. (2008) Vibrio cholera: Genomics and Molecular Biology. Caister Academic Press). Representative examples belonging to the heat-labile enterotoxin (LT) family protein include cholera toxin, which is a toxic factor A subunit (CTA) and a non-toxic factor B subunit (cholera toxin). B subunit; CTB) is a complex consisting of five. Among them, CTB has 5 monomers with a molecular weight of 12 kDa to form a 5-membered ring. AB5 toxin also includes heat-labile enterotoxin (ELT) derived from E. coli, and is similarly composed of toxin factor A subunit (LTA) and B subunit (LTB) pentamer (Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin.J Mol Biol. 230: 890-918). Similarly, Shigella, the causative agent of dysentery, is composed of A subunit and B subunit pentamer ( Silva, CJ, et al. (2017). Shiga Toxins: A Review of Structure, Mechanism, and Detection. Springer. ). As described above, the heat-labile enterotoxin (LT) family proteins have the same basic structure even though they are derived from various pathogens, and thus, expression of toxins and self-assembly into pentamers are achieved through similar mechanisms.

CTB, which is a representative example of a toxin protein belonging to the heat-labile enterotoxin (LT) family, possesses the ability to bind to the GM1 ganglioside receptor on the intestinal membrane surface (Cervin, J., et al. (2018) ).GM1 ganglioside-independent intoxication by Cholera toxin.PLoS pathogens, 14(2), e1006862.). Therefore, CTB is usefully used as a carrier function that promotes the absorption of chemically or genetically bound foreign antigens through the mucosa. In addition to these transporter functions, CTB is known to have various immunomodulatory functions (Royal, J., & Matoba, N. (2017). Therapeutic potential of cholera toxin B subunit for the treatment of inflammatory diseases of the mucosa.Toxins, 9 (12), 379.). The use of various useful proteins has been studied by utilizing these transporters and immune functions, and for this purpose, the production of a fusion protein that is mutually linked between CTB and a target protein has been reported. For example, insulin, insulin/GAD, and CTB fusion protein with full-length retrovirus NSP4 were expressed in transgenic potato plants, and the B chain of human insulin was expressed in transgenic tobacco plants (Arakawa, T., et al. (2001).Synthesis of a cholera toxin B subunit-rotavirus NSP4 fusion protein in potato.Plant cell reports, 20(4), 343-348.). Recently, it was confirmed that Ascaris suum As14 was successfully expressed in transgenic rice seeds (Nozoye, T., et al. (2009). Production of Ascaris suum As14 protein and its fusion protein with cholera toxin B subunit in rice seeds.Journal of Veterinary Medical Science, 71(7), 995-1000.).

However, although CTB-based fusion proteins are produced in an active form in the higher cells, prokaryotic systems such as E. coli have inherent limitations in their use due to the problem of protein folding. For example, when applied to the Japanese encephalitis virus vaccine antigen, the fusion protein is produced by insoluble precipitation, and despite the refolding process, which is a chemical solubility process, it cannot be produced as a complete pentamer, and a partial pentamer (fusion protein: (Harakuni, T., et al. (2005). Heteropentameric cholera toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal) Immune responses: a potential new strategy to target recombinant vaccine antigens to mucosal immune systems.Infection and immunity, 73(9), 5654-5665.). This is because, as described above, in prokaryotic cells such as E. coli that are genetically simple, there is essentially a limit to the protein folding function. Therefore, there is a functional limitation in inducing the folding structure and self-assembly of a protein derived from an animal-infected virus and a genetically modified fusion protein thereof. As a method to overcome this, a chemical protein refolding process can be used (Tamaki, Y., et al. (2016). Cholera toxin B subunit pentamer reassembled from Escherichia coli inclusion bodies for use in vaccination. Vaccine, 34(10), 1268-1274.). However, this requires enormous capital cost, and it is very difficult to ensure the quality of the final produced antigen and production efficiency as a pentameric substance.

Self-assembly of antigens into pentameric substances has a great relationship with increasing vaccine efficacy. In general, monomeric antigens are water-soluble, but have very low immunogenicity, and thus, in order to increase their effectiveness as a vaccine, it is necessary to form a multi-structure such as a multimer. Immunogenicity is the most important function of a vaccine. In order to increase this immunogenicity, the target antigen must be efficiently uptaken by specialized immune cells such as antigen presenting cells (APCs). Monomer water-soluble antigens rarely uptake APC, but when these monomers are made into structurally standardized complexes through self-assembly, it is known that the uptake to APC increases significantly and the vaccine efficacy increases (Jennings, GT, & Bachmann. , MF (2008).The coming of age of virus-like particle vaccines.Biological chemistry, 389(5), 521-536.; Zabel, F., et al. (2013). Virus-induced humoral immunity: on how B cell responses are initiated.Current opinion in virology, 3(3), 357-362.). Therefore, inducing self-assembly of target antigens into standardized nanoparticles can provide a major platform for vaccine design. However, self-assembly into such nanoparticles proceeds to a form close to a perfectly spherical shape under natural conditions. For example, in the case of the L1 protein, an envelope antigen of human papilloma virus (HPV) used as a female cervical cancer vaccine, 360 monomers are assembled and assembled into a virus-like particle (VLP) size (Wang, JW, & Roden, RB (2013).Virus-like particles for the prevention of human papillomavirus-associated malignancies.Expert review of vaccines, 12(2), 129-141.). In addition, in the case of a norovirus envelope protein used as a food poisoning enteritis vaccine, it is assembled into a VLP consisting of 180 monomers. Such self-assembly into VLPs is assembled via subunits (for example, trimers or pentamers) having a size smaller than this. However, there is no technology to stop such self-assembly at a specific stage (such as the pentagram stage) for use as a special-purpose vaccine or therapeutic agent, for example. As a method for implementing this, the target protein can be produced as a pentamer by fusing the target protein into a separate pentameric structure (scaffold). However, it is technically very difficult to produce a fusion protein as a water-soluble pentamer because when two different proteins are fused together, structural problems arise in terms of protein folding (Harakuni, T., et al. (2005). Heteropentameric cholera. toxin B subunit chimeric molecules genetically fused to a vaccine antigen induce systemic and mucosal immune responses: a potential new strategy to target recombinant vaccine antigens to mucosal immune systems.Infection and immunity, 73(9), 5654-5665.).

In order to solve this problem, in the present invention, B5 was used as a pentameric structure among the AB5 type toxin structures belonging to the heat-resistant toxin family, and a pentameric structure in order to efficiently produce a vaccine antigen whose formation into a pentameric structure is important. Phosphorus toxin protein and target antigen protein were fused and implemented. In addition, in order to successfully realize this in prokaryotic cells, an RNA-mediated folding function that promotes protein folding and self-assembly in prokaryotic cells was additionally combined (Choi, SI, et al. (2008). Protein solubility and folding enhancement by interaction with RNA.PLoS One, 3(7), e2677.). That is, the protein expression module is separated from the lysyl tRNA synthase N-terminal RNA binding domain (LysRS N-terminal appended RNA interacting domain; hereinafter RID), toxin-derived pentameric structural protein, triple fusion of the target protein. The present invention was completed by confirming that the produced recombinant protein was water-soluble to a high level in prokaryotic cells, the pentameric structure was well formed, and the ability to form neutralizing antibodies was excellent.

An object of the present invention is a recombinant expression vector capable of expressing in a form capable of increasing immunogenicity as well as producing a target protein in a water-soluble manner, which is difficult to express in water-soluble in prokaryotic cells; Host cells transformed with the expression vector; And a recombinant fusion protein in a pentameric form expressed in the host cell.

Another object of the present invention is to provide a method for producing a recombinant fusion protein in a pentameric form using the expression vector and host cells.

Another object of the present invention is to provide a virus diagnostic composition or vaccine composition comprising the recombinant fusion protein.

The present invention is characterized by the mutual fusion of i) an RNA binding domain (LysRS N-terminal appended RNA interacting domain, RID), ii) a pentameric toxin protein, and iii) a protein of interest. The present invention was completed by verifying that not only can produce a polymer, but also that the purified pentameric protein exhibits excellent neutralization against the virus of interest.

Accordingly, the present invention is an N-terminal RNA binding domain isolated from lysyl tRNA synthetase (LysRS N-terminal appended RNA interacting domain; RID); Pentameric toxin protein; And it provides a recombinant fusion protein expression vector comprising a polynucleotide encoding the protein of interest.

In the present specification, "target protein" refers to a protein that a person skilled in the art wants to produce in large quantities, and refers to a protein that can be expressed in a host cell by inserting a polynucleotide encoding the protein into a recombinant expression vector. This is a viral antigen protein that induces an immune response.

In the present invention, the virus is a virus having a spherical icosahedral capsid structure, and the icosahedron is a structure made by gathering 20 equilateral triangles, which are subunits, and 12 vertices are largely two-fold axis, 3 There may be a three-fold axis and a five-fold axis, and a subunit may be formed of a combination of pentagons or hexagons depending on the axis.

Accordingly, the target protein of the present invention may be a viral antigen protein that forms pentameric or hexamer as subunits in the self-assembly process. In general, it can be applied to the pentameric constituent antigens of all enveloped or non-enveloped viruses constituting the icosahedron.

Examples of enveloped viruses having a lipid membrane may include Herpesviridae and Hepadnaviridae viruses having a DNA genotype, and Flaviviridae viruses having an RNA genotype. Can be included. In a specific embodiment of the present invention, a recombinant vector and a recombinant protein are prepared using Dengue virus (hereinafter DV) and Japanese Encephalitis virus (hereinafter JEV) belonging to the flavivirus genus, and water-soluble in prokaryotic cells. Expression, formation of a pentameric structure, and the immune ability of neutralizing antibodies were confirmed.

In a specific embodiment of the present invention, the ED3 (E protein domain 3) of the dengue virus is assembled into a pentameric body and directly binds to a receptor of a host cell, and viral fusion and a viral gene to the host cell It plays a very important role in intracellular infection through transmission (Zhang, X., et al. (2017). Structures and functions of the envelope glycoprotein in flavivirus infections. Viruses, 9(11), 338.). ED3 of the present invention may be represented by the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. In addition, the recombinant vector of the present invention may include a polynucleotide of SEQ ID NO: 2 encoding SEQ ID NO: 1 and a homologue thereof. Specifically, it may include a nucleotide sequence having sequence homology of 70% or more, more preferably 80% or more, and most preferably 90% or more, respectively, with the nucleotide sequence of SEQ ID NO: 2.

[SEQ ID NO: 1]

Dengue virus ED3 amino acid sequence

KGVSYVMCTGSFKLEKEVAETQHGTVLVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAEPPFGESYLVVGAGEKALKLSWFKKG

[SEQ ID NO: 2]

Nucleotide sequence encoding dengue virus ED3

aaaggcgtctcctacgtaatgtgtaccggcagcttcaagttagaaaaagaggttgccgagacccagcatgggaccgtacttgtccaggtgaagtacgagggcactgatgcaccctgtaaaatccccttctccagtcaagacgagaagggggtgacacaaaatggtcgtctgatcacagcgaaccctatcgtgacggataaggaaaagcccgttaacatcgaagcggaacctccgtttggcgaaagctatttagtagtaggggcgggggagaaagctctgaagttatcgtggttcaagaaaggc

In a specific embodiment of the present invention, the JEV of the Japanese encephalitis virus may be represented by the amino acid sequence of SEQ ID NO: 3, but is not limited thereto. In addition, the recombinant vector of the present invention may include the polynucleotide of SEQ ID NO: 4 encoding SEQ ID NO: 3 and homologs thereof. Specifically, it may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, and most preferably 90% or more, respectively, with the nucleotide sequence of SEQ ID NO: 4.

[SEQ ID NO: 3]

JEV amino acid sequence of Japanese encephalitis virus

kgttygmctekfsfaknpadtghgtvvielsysgsdgpckipivsvaslndmtpvgrlvtvnpfvatssanskvlvemeppfgdsyivvgrgdkqinhhwhkag

[SEQ ID NO: 4]

JEV nucleotide sequence of Japanese encephalitis virus

aaaggcacaacctatggcatgtgcacagaaaaattctcgttcgcgaaaaatccggcggacactggtcacggaacagttgtcattgaactttcctactctgggagtgatggcccttgcaaaattccgattgtctccgttgcgagcctcaatgacatgacccccgtcgggcggctggtgacagtgaaccccttcgtcgcgacttccagcgccaactcaaaggtgctagtcgagatggaaccccccttcggagactcctacatcgtagttggaaggggagacaagcagattaaccaccattggcacaaggctgga

In the present invention, the non-enveloped virus (non-enveloped or naked virus) composed of a viral envelope protein without a lipid membrane may include a virus of the genus Adenovirus (Adenoviridae) and a genus parvovirus (Pavoviridae) having a DNA genotype, and RNA Genotype Reoviridae, Picornavirus genus (Picornaviridae), Calicivirus genus (Caliciviridae) viruses may be included. In a specific embodiment of the present invention, a recombinant vector and a recombinant protein are prepared using the capsid protein of a foot-and-mouth disease virus (FMDV) belonging to the genus Picornaviridae, and then in prokaryotic cells. The water-soluble expression, formation of a pentameric structure, and the immune ability of the neutralizing antibody were confirmed. Foot-and-mouth disease virus envelope proteins are composed of a total of 180 proteins, consisting of a total of 60 proteins VP1, VP0, and VP3, forming a single icosahedron. Among the three different constituent antigens (VP1, VP0, VP3), the assembly of the pentamer is composed of the VP1 protein.

In a specific embodiment of the present invention, VP1 of the foot-and-mouth disease virus may be represented by the amino acid sequence of SEQ ID NO: 5, but is not limited thereto. In addition, the recombinant vector of the present invention may include the polynucleotide of SEQ ID NO: 6 encoding SEQ ID NO: 5 and homologs thereof. Specifically, it may include a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, and most preferably 90% or more, respectively, with the nucleotide sequence of SEQ ID NO: 6.

[SEQ ID NO: 5]

Foot-and-mouth disease virus VP1 amino acid sequence

ttttgesadpvtttvenyggetqtarrlhtdvafvldrfvkltqpkstqtldlmqipshtlvgallrsatyyfsdlevalvhtgpvtwvpngapktalnnhtnptayqkqpitrlalpytaphrvlstvyngkttygeessrrgdlaalarrvnnrlptsfnygavkadtitellirmkraeqrpnnrlptsfnygavkadtitellirmkraetyrp

[SEQ ID NO: 6]

Foot-and-mouth disease virus VP1 nucleotide sequence

accactaccaccggtgaaagcgcagacccggtaactactaccgttgaaaactacgggggcgaaacccaaacggcgcgtcgtctgcatactgacgtagcgttcgtgctggaccgcttcgttaaactgacccaaccgaaatcaactcagacacttgatttaatgcagatcccttcccacaccctggtgggtgctctattgcgatctgccacctactacttcagcgatctggaagttgccctggtgcataccgggccggttacctgggttccgaacggcgctccgaaaaccgcactcaacaaccacaccaatccaacagcatatcagaaacagccgatcacccgtctggcgctgccttatactgccccgcatcgtgttctgagcacggtgtacaacggtaaaacgacctatggcgaagaatcgtctcgtcgtggtgacctggcggcgttggcacgtcgcgtgaataaccgcctgccgacctctttcaactacggtgcggtaaaagccgataccatcaccgaactgctgatccgtatgaaacgtgcggaaacctactgcccgcgcccgttgctggcactggataccacgcaggatcgccgtaaacaaaaaatcatcgcaccggaaaaacagatgatc

In the present specification, "pentameric toxin protein" refers to cholera toxin B subunit (CTB) protein, heat-labile enterotoxin B subunit (LTB), and Shiga toxin B subunit (Shiga -toxin B subunit) may be selected from the group consisting of proteins or fragments thereof, but can be used without limitation as long as the activity to induce pentameric structure is maintained. The CTB is very similar to the LTB secreted from E. coli (E.coli) or Bacillus cereus (Bacillus cereus) a structure or mechanism of toxicity (Sixma, TK, et al. (1993). Refined structure of Escherichia coli heat-labile enterotoxin, a close relative of cholera toxin.Journal of molecular biology, 230(3), 890-918.). In addition, it is structurally very similar to the Shiga toxin B subunit derived from Shigella, a heterogeneous causative bacterium ( Silva, CJ, et al. (2017). Shiga Toxins: A Review of Structure, Mechanism, and Detection. Springer. ). Therefore, it can be easily applied to CTB, LTB, Shiga toxin B subunit, and other similar toxins for pentomer-forming antigen fusion in the present invention.

In a specific embodiment of the present invention, it was confirmed that the antigenic protein fused with CTB and LTB forms a pentameric structure. In the present invention, the CTB protein may be a protein or fragment formed by adding, deleting or substituting some amino acid residues in the amino acid sequence of SEQ ID NO: 7. In the present invention, the LTB protein may be a protein or fragment formed by adding, deleting or substituting some amino acid residues in the amino acid sequence of SEQ ID NO: 9.

[SEQ ID NO: 7]

CTB amino acid sequence

TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN

[SEQ ID NO: 8]

CTB nucleotide sequence

actccgcagaacattacggacctgtgtgcggagtatcataatacgcagattcacactttgaatgacaagattttttcatatacggagtcattagctggtaaacgtgaaatggcaattatcacttttaaaaatggtgcgacgttccaggtggaagttccgggcagtcagcatattgatagtcagaaaaaagccatcgaacgtatgaaggataccttgcgtattgcgtacttaaccgaggctaaagtcgagaaattatgtgtctggaataataagaccccacatgccattgctgcgatttcgatggccaat

[SEQ ID NO: 9]

LTB amino acid sequence

Apqtitelcseyrntqiytindkilsytesmagkremviitfksgetfqvevpgsqhidsqkkaiermkdtlrityltetkidklcvwnnktpnsiaaismkn

[SEQ ID NO: 10]

LTB nucleotide sequence

gctccccagactattacagaactatgtgttcggaatatcgcaacacacaaatatatacgataaatgacaagatactatcatatacggaatcgatggcaggcaaaagagaaatggttatcattacatttaagagcggcgaaacatttcaggtcgagaagtcccggggaattgagatagatagattgagatgagatagatagattgagaaa

In the present specification, "RNA interacting domain (RID)" or "LysRS N-terminal appended RNA interacting domain (RID)" or "LysRS RNA binding domain isolated from lysyl tRNA synthase" "" means a unique N-terminal extension site involved in the interaction between RNA and other proteins of LysRS. In a specific embodiment of the present invention, the RID may be a protein or fragment formed by adding, deleting or substituting some amino acid residues in the amino acid sequence of SEQ ID NO: 11. In the present invention, the RID may be used without limitation as long as it has the ability to interact with RNA and maintains an activity capable of enhancing the expression efficiency and water solubility of the target protein.

[SEQ ID NO: 11]

hRID amino acid sequence

MSEQHAQAAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

[SEQ ID NO: 12]

hRID nucleotide sequence

atgtctgaacaacacgcacaggcggccgtgcaggcggccgaggtgaaagtggatggcagcgagccgaaactgagcaagaatgagctgaagagacgcctgaaagctgagaagaaagtagcagagaaggaggccaaacagaaagaggaggccaaacagaaagagccgaggaggagcccgtgagggaggagccactgctgaggggagccactgct

The term "expression vector" as used herein is a linear or circular DNA molecule composed of a fragment encoding a protein of interest operably linked to an additional fragment provided for transcription of the expression vector. Such additional fragments include promoter and terminator sequences. Expression vectors also include one or more origins of replication, one or more selectable markers, and the like. Expression vectors are generally derived from plasmid or viral DNA or contain elements of both.

The term “operably linked” as used herein refers to the arrangement of fragments to act to initiate transcription at the promoter and proceed to the termination code through the coding sequence.

The vector for producing a recombinant fusion protein of the present invention may include a polynucleotide encoding the peptide of SEQ ID NO: 11 for enhancing water-soluble expression and expression efficiency of the target protein.

In a preferred embodiment of the present invention, the polynucleotide encoding the peptide of SEQ ID NO: 11 may be a sequence represented by SEQ ID NO: 12.

The recombinant fusion protein expression vector of the present invention may include a polynucleotide encoding the peptide of SEQ ID NO: 13 for enhancing the expression of the target protein in a pentameric form.

In a preferred embodiment of the present invention, the polynucleotide encoding the peptide of SEQ ID NO: 13 may be a sequence represented by SEQ ID NO: 14.

In a preferred embodiment of the present invention, the recombinant fusion protein expression vector comprises 1 to 6 polynucleotides encoding histidine; And a polynucleotide encoding a protein cleavage enzyme recognition site.

In the recombinant fusion protein expression vector of the present invention, the protein cleavage enzyme may be TEV.

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 regardless of the genome of the host cell or integrated into the genome of the host cell.

The present invention also includes an N-terminal RNA binding domain isolated from lysyl tRNA synthetase (LysRS N-terminal appended RNA interacting domain; RID); Pentameric toxin protein; And it provides a host cell transformed with a recombinant fusion protein expression vector comprising a polynucleotide encoding the desired protein.

As used herein, the term "transformation" or "introduction" means that DNA is introduced into a host so that the DNA becomes replicable either as an extrachromosomal factor or by completion of chromosomal integration. Methods of transforming the expression vector according to the present invention include electroporation, calcium phosphate (CaPO ₄ ), calcium chloride (CaCl ₂ ), microinjection, polyethylene glycol (PEG), and DEAE-dex. Tran method, cationic liposome method, or lithium acetate-DMSO method may be included, but are not limited thereto.

In the host cell according to the present invention, the host cell is preferably a host cell having high DNA introduction efficiency and high expression efficiency of the introduced DNA, and all microorganisms including prokaryotic and eukaryotic can be used. In general, folding of proteins or self-assembly of monomers is much more difficult in case of relatively simple prokaryotic cells compared to eukaryotic cells. In particular, in the case of virus-derived antigens that infect humans, animals, etc., which are targets of the vaccine, folding or self-assembly is much easier in eukaryotic cells such as human, animal-derived higher cells or yeast. In contrast, as described above, in the case of E. coli, folding/self-assembly of antigens is technically very difficult, and therefore, there is a limitation in the process requiring refolding of an amorphous inclusion body that is not folded (misfolding). Therefore, in E. coli, which is a representative example of prokaryotic cells used in the present invention, folding into pentagrams and self-assembly implemented by overcoming the limitations of the existing technology can be very easily implemented in eukaryotic cells such as humans, animals, insect cells, and yeast. It's self-evident. Therefore, the host cells used in the present invention include bacteria {Escherichia genus bacteria; Bacteria of the genus Bacillus; Bacteria of the genus Pseudomonas; Including lactic acid bacteria, etc.} as well as yeast (bakers yeast such as Sacharomyceses cerevisiae, S. pombe; methylotrophic yeast such as Pichia pastoris); Animal cells; Insect cells; It may be selected from the group consisting of plant cells, and preferably, the host cell may be E. coli.

The present invention also provides a method for producing a recombinant fusion protein with improved water-soluble expression efficiency. The method for producing the recombinant fusion protein

(a) N-terminal appended RNA interacting domain (RID) isolated from lysyl tRNA synthetase; Pentameric toxin protein; And preparing an expression vector comprising a polynucleotide encoding a protein of interest;

(b) preparing a transformant by introducing the expression vector into a host cell; And

(c) inducing the expression of the RID-CTB-target protein fusion protein by culturing the transformant; It may include.

In the method for producing a recombinant fusion protein according to the present invention, the host cell is preferably a host cell having high DNA introduction efficiency and high expression efficiency of the introduced DNA, and all microorganisms including prokaryotic and eukaryotic can be used. The host cells are bacteria of the genus Escherichia; Bacillus Bacillus bacteria; Bacteria of the genus Pseudomonas; Lactobacillus; leaven; Animal cells; And it may be selected from the group consisting of insect cells, preferably, the host cell may be E. coli (E. coli).

The present invention also includes an N-terminal RNA binding domain isolated from lysyl tRNA synthetase (LysRS N-terminal appended RNA interacting domain; RID); Cholera toxin B subunit (CTB) protein; And it provides a recombinant fusion protein comprising a protein of interest.

As used herein, the term "fusion protein" or "recombinant protein" refers to a protein in which another protein is linked or a different amino acid sequence is added to the N-terminus or C-terminus of the original target protein sequence. Means.

As used herein, the term “protein” is used interchangeably with “peptide” or “polypeptide”, and refers to a polymer of amino acid residues, for example, as commonly found in proteins in nature.

In one embodiment of the present invention, the recombinant fusion protein refers to a RID-CTB-target protein fusion protein, and the recombinant fusion protein may be expressed in a pentameric form.

In a preferred embodiment of the present invention, the recombinant fusion protein may be represented by the amino acid sequence of SEQ ID NO: 9, but is not limited thereto.

The present invention also provides a composition for diagnosing viral infection comprising the expression vector or the recombinant fusion protein.

In the present invention, "diagnosis" means confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, the diagnosis is to determine whether or not a virus has developed or is likely to develop.

The composition for diagnosing viral infection may further include reagents generally used in the art used for immunological analysis, as well as the recombinant fusion protein including the viral antigen protein. Such reagents include, but are not limited to, a labeling substance capable of generating a detectable signal, a solubilizing agent, a detergent, a buffering agent, and a stabilizer. When the labeling substance is an enzyme, a substrate capable of measuring enzyme activity and a reaction terminator may be included.

The present invention also provides a vaccine composition for enhancing immunity comprising the expression vector or the recombinant fusion protein.

The vaccine composition according to the present invention may contain the expression vector or the recombinant fusion protein alone or may be formulated in a suitable form with a pharmaceutically acceptable carrier, and may further contain an excipient or diluent. In the above, "pharmaceutically acceptable" refers to a non-toxic composition that is physiologically acceptable and does not cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness when administered to humans.

The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery substances used for oral administration of the peptide preparation. In addition, the carrier for parenteral administration may include water, suitable oils, saline, aqueous glucose and glycol, and the like, and may further include stabilizers and preservatives. Suitable stabilizers are antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, and the like in addition to the above components. Other pharmaceutically acceptable carriers and agents may be referred to as those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed, Mack Publishing Company, Easton, PA, 1995).

The vaccine composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration Can be

The vaccine composition of the present invention may be formulated as a formulation for oral administration or parenteral administration according to the route of administration as described above.

In the case of a formulation for oral administration, the composition of the present invention may be formulated using a method known in the art as a powder, granule, tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc. I can. For example, in oral preparations, tablets or dragees can be obtained by blending the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing it into a granule mixture. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches, including corn starch, wheat starch, rice starch and potato starch, and the like, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose, gelatin, polyvinylpyrrolidone, and the like may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant.

Furthermore, the vaccine composition of the present invention may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative. In the case of a formulation for parenteral administration, it may be formulated in the form of injections, creams, lotions, ointments for external use, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed, Mack Publishing Company, Easton, PA, 1995, a formula generally known for all pharmaceutical chemistry.

The total effective amount of the composition of the present invention may be administered to a patient in a single dose, and may be administered by a fractionated treatment protocol that is administered for a long period in multiple doses. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the severity of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be about 001 μg to 10,000 mg, most preferably 0.1 mg to 500 mg per 1 kg of the patient's body weight per day. However, the dosage of the pharmaceutical composition is determined by considering various factors such as the age, weight, health status, sex, disease severity, diet and excretion rate of the patient, as well as the formulation method, route of administration and number of treatments. Therefore, considering these points, those of ordinary skill in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration, and method of administration as long as it exhibits the effects of the present invention.

In the present invention, CTB was used as a scaffold capable of inducing a standardized interaction between monomers, and all these problems were solved through fusion of three heterologous proteins. Surprisingly, this combination of domains induces folding of foreign proteins and multi-structure complexes (multimers) even in cells such as Escherichia coli, thereby greatly improving the water solubility and the efficiency of generating pentamers. Through this, the efficiency of pentagram formation was improved by more than 200-300% compared to the existing technology through plant cells, which are higher cells, even though they were actually expressed in the E. coli system (Kim, TG, et al. (2010). Cholera toxin B subunit-domain). III of dengue virus envelope glycoprotein E fusion protein production in transgenic plants.Protein expression and purification, 74(2), 236-241.). In addition, as a result of inoculating the expressed protein into experimental mice and analyzing the neutralizing antibody titer, it was proved that the protective effect was improved by 200-300% or more compared to the recombinant protein vaccine not fused with cholera toxin B.

According to the recombinant expression vector of the present invention and the method for producing a recombinant fusion protein using the same, it is possible to produce an antigenic protein in high yield by greatly improving the expression amount and water solubility of the recombinant protein in cells, as well as structurally elaborate pentameric antigenic protein. Can produce.

In addition, the viral vaccine comprising the fusion protein in the form of a pentameric form of the present invention can greatly improve immunogenicity due to the formation of an elaborate pentameric body.

FIG. 1 is a schematic diagram of a spherical icosahedral shell virus (A) and a structure modeled through UCSF chimera (B), showing a twofold axis, a threefold axis, and a fivefold axis that occur when the icosahedron is formed. axis) and its subunits.
Figure 2 shows a schematic diagram of a pGE-hRID (human-derived RID) vector configuration and pentameric CTB-DV1ED3 for expression of the recombinant protein of the present invention.
3 is a result of confirming the expression amount and water solubility of the recombinant ED3 protein expressed according to an embodiment of the present invention by SDS-PAGE. M is the marker, T is the total cell lysate solution, S is the soluble fraction after centrifugation, P is the pellet fraction after centrifugation.In each case, the left panel is the expression result of CTB-DV1ED3 without recombination of hRID, and the right panel is This is the result of expression of hRID-CTB-DV1ED3 in which hRID has been recombined.
4 is a result of confirming the fraction by SDS-PAGE after purifying the recombinant protein expressed according to an embodiment of the present invention through nickel-affinity chromatography. (A) is the result of confirming whether or not the protein is purified by performing SDS-PAGE for each fraction and the fraction in which the purified protein is present, and the number on the figure indicates the fraction number divided during purification. (B) is the result of calculating the total amount of the recombinant protein obtained by comparing the BSA (1 mg/ml) and the band density after purification. Considering that the concentration of this recombinant protein is very high, it was first diluted 1/4 and loaded. The average concentration calculated from the band density was 4.803 μg/μl and the total volume was about 4 ml, so a total of 19.212 mg of recombinant protein was obtained through 500 ml culture.
5 is a result of confirming the formation of pentamers of the expressed and purified recombinant protein according to an embodiment of the present invention through SDS-PAGE. This was confirmed by minimizing denaturation of the recombinant protein by loading the sample without boiling and without DTT. Since hRID has the property of making the size of the fusion protein appear larger, a sample cut using TEV protease was also confirmed to rule out this.
6 is a result of calculating the correct size through size-exclusion chromatography (SEC) to determine whether a pentamer is formed of the expressed and purified recombinant protein according to an embodiment of the present invention. (A) is the SEC result of hRID-CTB-DV1ED3, and (B) is the SEC result of CTB-DV1ED3 cut hRID using TEV protein cleavage enzyme. As a result of quantifying and calculating the peak obtained through SEC, the size of the peak appearing in the recombinant protein by drawing a trend line through the calibration sample was calculated (Kav = partition coefficient, Vo = column void volume (8.67), Ve = elution volume. , and Vc = geometric column volume (25)).
7 is a diagram showing an experimental group and an advanced schedule configured when inoculating a mouse with a recombinant protein expressed and purified according to an embodiment of the present invention.
8 is an ELISA result confirming the reaction with the wild-type dengue virus with serum obtained from group 1 (hRID-DV1ED3) and group 2 (hRID-CTB-DV1ED3) according to an embodiment of the present invention. Each serum was initially diluted 1/200 and used, and the virus was coated with ^{10 4 PFU per well.}
9 is a result of NT assay for wild-type dengue virus 1 with serum obtained through a mouse experiment. The NT titer is designated as the previous dilution multiple and the subsequent dilution multiple before and after the point where the number of flaks decreased by 50% compared to the control group, and [NT titer = previous dilution multiple + (50% of the number of flaks in the control group-the number of flaks in the previous dilution multiple) *(After dilution multiple-Previous dilution multiple)/(After dilution multiple-Number of flaks of previous dilution multiple)]. The detection limit is 20.
FIG. 10 is a result of confirming by SDS-PAGE the vector composition (A) for expression of the recombinant Japanese encephalitis virus (JEV) antigen protein according to an embodiment of the present invention, and the expression amount and water solubility of the expressed recombinant protein (B ). M is a marker, T is total cell lysate solution, S is soluble fraction after centrifugation, P is pellet fraction after centrifugation, the left figure is the expression result of hRID-CTB-JEVEDEDIII without GPGP recombination, the right figure is GPGP Is the result of the recombinant hRID-CTB-GPGP-JEVEDEDIII expression.
11 is a result of confirming the fraction by SDS-PAGE after purifying the Japanese encephalitis virus recombinant protein expressed according to an embodiment of the present invention through nickel-affinity chromatography. The number above the figure means the fraction number divided during purification, the left figure is the result of hRID-CTB-JEVEDEDIII in which GPGP is not recombined, and the right figure is the result of hRID-CTB-GPGP-JEVEDEDIII in which GPGP is recombined.
12 is a result of confirming the formation of pentamers of the Japanese encephalitis virus recombinant protein expressed and purified according to an embodiment of the present invention through SDS-PAGE. The sample was loaded without boiling and without DTT, thereby minimizing denaturation of the recombinant protein to confirm the formation of pentamers.
13 is a result of confirming the composition of the vector for the expression of the foot-and-mouth disease virus (FMDV) recombinant antigen protein according to an embodiment of the present invention (A) and the expression amount and water solubility of the expressed recombinant protein by SDS-PAGE (B) . M is the marker, T is the total cell lysate solution, S is the soluble fraction after centrifugation, P is the pellet fraction after centrifugation, and S'is the S fraction is loaded without DTT treatment and without boiling. The left figure shows the expression result of CTB-VP1 without recombination of hRID, the middle figure shows the expression result of hRID-CTB-VP1 without recombination of hRID and GPGP, and the right figure shows the recombination of both hRID and GPGP. These are the hRID-CTB-GPGP-VP1 expression results.
14 is a result of confirming the fraction by SDS-PAGE after purifying the recombinant foot-and-mouth disease virus protein expressed according to an embodiment of the present invention through nickel-affinity chromatography. The number above the figure means the fraction number divided during purification, the left figure is the result of hRID-CTB-VP1 in which GPGP is not recombined, and the right figure is the result of hRID-CTB-GPGP-VP1 in which GPGP is recombined.
15 is a result of confirming the formation of pentamers of the expressed and purified foot-and-mouth disease virus recombinant protein according to an embodiment of the present invention through SDS-PAGE. The sample was loaded without boiling and DTT was not added to minimize denaturation of the recombinant protein to confirm the formation of pentamers.
16 is a result of confirming the formation of pentamers of the LTB fusion protein according to an embodiment of the present invention through SDS-PAGE. The sample was loaded without boiling and DTT was not added to minimize denaturation of the recombinant protein to confirm the formation of pentamers. (A) is the result of hRID-LTB and hRID-LTB-FMDV VP1, (B) is the result of hRID-LTB-JEVEDIII.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrative purposes only, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not construed as being limited by these examples.

[Example 1]

Construction of recombinant expression vector

The pGE-hRID expression vector was used as the protein expression vector (YANG, Seung Won, et al. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.The FASEB Journal, 2018, fj. 201700747RR.) . The expression of pGE-hRID is regulated by the T7 promoter, and TEV (tobacco etch virus) protease site (ENLYFQ) was inserted to cleave the fusion protein (hRID) if necessary. In addition, a 6X His tag was inserted to perform nickel-affinity chromatography, and the MCS (Multiple Cloning Site) composed of Kpn1-BamH1-EcoRV-Sal1-Hind3 was digested with Kpn1 and Hind3, and the target protein was fused with CTB. The sequence was inserted (Fig. 2A). In order to compare the degree of water-soluble expression of the target protein in E. coli according to the presence or absence of hRID, the vector into which the hRID was inserted (1) and the vector from which the hRID was removed (2) were used (FIG. 2B).

Specifically, hRID by inserting the gene sequence encoding CTB (SEQ ID NO: 8) and Dengue virus serotype 1 (DV1) ED3 encoding gene sequence (SEQ ID NO: 2) using Kpn1 and Hind3 restriction enzymes into the pGE-hRID plasmid. A recombinant vector in the form of -CTB-DV1ED3 was prepared, and a recombinant vector in the form of CTB-DV1ED3 from which the hRID sequence (SEQ ID NO: 12) was removed was prepared as a control vector. In this vector, promoter activation is regulated by IPTG.

[Example 2]

Confirmation of water-soluble expression of the recombinant protein of the present invention

의 조건에서 배양하였으며, 대장균의 OD₆₀₀ 값이 0.5 이상이 되면 T7 promoter를 활성화 시키기 위해서 IPTG를 1 mM 수준으로 넣어주고, 단백질이 충분히 생산될 수 있도록, IPTG를 넣어준 이후부터 20

에서 5시간 정도를 배양하였다. 충분히 배양된 대장균은 원심분리하여 상등액을 제거한 후에 보관하였다. 그 다음, 대장균 수확물에 A buffer[50mM Tris-Cl(pH7.5), 300mM NaCl, 10% glycerol, 5mM imidazole] 10ml을 넣은 뒤 초음파 분쇄를 하여, 용해물(lysate)을 만들었다. 그 후, 상기 용해물을 SDS-PAGE로 분석하였다.In Example 1 The prepared protein expression vector was transformed into SHuffle® T7 competent cells (New England Biolabs #C3026) and cultured. The transformed E. coli was cultured in LB medium containing 50 μg/ml of ampicillin. Incubation temperature is 37

When the OD ₆₀₀ value of E. coli exceeds 0.5, IPTG was added at 1 mM level to activate the T7 promoter, and IPTG was added to allow sufficient protein production.

Incubated for about 5 hours. The sufficiently cultured E. coli was stored after centrifugation to remove the supernatant. Then, 10 ml of A buffer [50mM Tris-Cl (pH7.5), 300mM NaCl, 10% glycerol, 5mM imidazole] was added to the harvest of E. coli, followed by ultrasonic grinding to prepare a lysate. Then, the lysate was analyzed by SDS-PAGE.

As a result, about half of the total protein expression amount of the recombinant protein of the present invention fused with hRID was expressed in a water-soluble manner. On the other hand, in the control without hRID fusion (CTB-DV1ED3-direct), most of the proteins were expressed in an insoluble form (FIG. 3).

Through this, it was found that even in the case of a recombinant protein fused with CTB, the efficiency of water-soluble expression of the recombinant protein can be greatly increased through hRID fusion.

[Example 3]

Purification and expression yield confirmation of the recombinant protein of the present invention

The recombinant protein whose expression was confirmed in Example 2 was produced by scale-up culture to 500ml. Since the recombinant protein has a 6x His tag, it can be effectively separated through a Ni-column. Therefore, a soluble fraction subjected to ultrasonic grinding and centrifugation was loaded onto the Ni column, and the protein was eluted according to a linear gradient from 10 mM to 300 mM of imidazole. The composition of the buffer used for purification is as follows. A buffer[50mM Tris-Cl(pH7.5), 300mM NaCl, 10% glycerol, 5mM imidazole], B buffer[50mM Tris-Cl(pH7.5), 300mM NaCl, 10% glycerol, 300mM imidazole].

Next, all the fractions obtained through nickel-affinity chromatography were analyzed by SDS-PAGE, of which only the fractions containing the recombinant protein, that is, fractions 16 to 24 in FIG. 4A, were mixed. After collecting, it was concentrated using a centrifugal filter (Centriprep; EMD Millipore, Billerica, MA, USA), and dialysis with C buffer [50mM Tris-Cl (pH 7.5), 300mM NaCl, 0.1mM EDTA] Kept. The concentration of purified pure hRID-CTB-DV1ED3 protein was measured by comparing the band density with BSA whose concentration was already known through SDS-PAGE.

As a result, as shown in [Fig. 4B], the amount of recombinant protein expressed and purified through 500ml of E. coli was calculated to be a total of 19.212mg.

[Example 4]

Confirmation of the pentameric structure of the recombinant protein of the present invention through SDS-PAGE

SDS-PAGE was first performed to confirm whether the recombinant protein obtained through the purification of Example 3 had a pentameric structure. Unlike the previous SDS-PAGE, since the structure of the protein must be maintained in an undenaturation state, a loading buffer without DTT was used, and the boiling process was omitted.

As a result, as a result of comparing and measuring the band density, it can be seen that most of the proteins are formed as pentamers even though SDS is included (Fig. 5), which in fact can be estimated that a larger proportion of proteins form pentamers. I can. In addition, when recalling that the physical properties of most proteins are mutually bonded to SDS, this result suggests that the formed pentamer is self-assembled into an ordered structure that is structurally very stable.

[Example 5]

Verification of the pentameric structure of the recombinant protein of the present invention through size exclusion chromatography

In order to reliably verify the formation of the pentameric structure of the recombinant protein identified in Example 4, size exclusion chromatography (SEC) was performed using Superdex-200 Increase column (GE Healthcare). Calibration was performed with eggplant marker protein. The composition of the buffer used at this time was [50mM Tris-Cl (pH 7.5), 300mM NaCl], and was separated through a Superdex-200 analytical gel-filtration column (GE Healthcare). All fractions were analyzed by SDS-PAGE, and only fractions containing proteins corresponding to the pentagram size were collected and used.

As a result, the main peak of the recombinant protein appeared near the ferritin peak (440 kDa) used for calibration. As a result of calculating this through the elution volume and partition coefficient (K _av ), it was found to have a size of about 476kDa, which is due to the size shift property of hRID (FIG. 6A). In fact, in the SEC result of CTB-DV1ED3 cleaved hRID, a peak appeared at about 128 kDa, which can be confirmed that the pentameric structure was accurately formed by considering that the size of the CTB-DV1ED3 monomer was about 25 kDa (Fig. 6B).

[Example 6]

Antibody acquisition and measurement of neutralizing antibody titer through ELISA

<6-1> Animal experiment to obtain serum for the recombinant protein of the present invention

에 보관하였다. 상기 모든 동물실험은 연세대학교 동물실험윤리위원회의 심의를 거쳐 진행되었다(IACUC-A-201701-108-03, IACUC-A-201709-352-02).In order to confirm the immunogenicity when the recombinant protein, which has been expressed, purified, and verified, is inoculated into a mouse, and whether the antibody has a neutralizing ability, an animal experiment was first conducted to obtain an antibody. Animal experiments were conducted on the same schedule as in [Fig. 7], and blood samples were obtained through orbital blood collection after inoculation at intervals of 2 weeks. Specifically, the mice used in the experiment were all 6-week-old female BALB/c species (Orient Bio, South Korea), and 20 μg of antigen protein was inoculated three times at 2-week intervals through subcutaneous injection (SQ). As an adjuvant, alum (Imject Alum adjuvant; Thermo Fisher Scientific) was mixed and used so that the volume ratio with the final antigen protein was 1:3 (antigen protein: alum). Two weeks after each inoculation, blood samples were obtained through orbital blood collection, and the obtained blood samples were separated by centrifugation to separate only serum and aliquoted to -20

Stored in. All of the above animal experiments were conducted after deliberation by the Animal Experimental Ethics Committee of Yonsei University (IACUC-A-201701-108-03, IACUC-A-201709-352-02).

<6-2> Measurement of serum antibody titer

에서 하루 동안 반응시켜 코팅했다. 그 후, TBST로 워싱하고 블로킹을 위해 TBST에 1% BSA를 섞어 한 시간 동안 실온에서 반응시켰다. 다시 워싱 후 초기 1/200 희석한 후 2배 단계 희석(2-fold serial dilution)한 100μl의 혈청샘플들을 각 웰마다 넣어준 후 실온에서 두 시간 반응시켰다. 마찬가지로 워싱 후에, 1:10,000으로 희석한 HRP conjugated goat anti-mouse IgG(Sigma Aldrich)를 100μl씩 넣어 실온에서 한 시간 반응시켰다. 마지막으로 워싱 단계를 거친 후에 3,3',5,5'-tetramethybenzidine(TMB) solution(BD bio sciences)을 각 웰마다 100μl씩 넣고 실온에서 30분 반응시켰고, 그 후 2N H₂SO₄ 50μl를 웰마다 넣어 반응을 멈추게 한 후 microplate reader(FLUOstar Optima; BMG Labtech)를 통해 OD₄₅₀ 값을 측정하였다. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the antibody titer of serum obtained through animal experiments. Dengue virus (NCCP41507, National Culture Collection for Pathogen, South Korea) was added to a 96-well Nunc plate (Thermo Fisher Scientific) at ^{a concentration of 10 4} PFU/well.

It was coated by reacting for a day at. Thereafter, washing with TBST, 1% BSA was mixed with TBST for blocking, and reacted at room temperature for one hour. After washing again, an initial 1/200 dilution was performed, and then 100 μl of serum samples subjected to 2-fold serial dilution were added to each well, and then reacted at room temperature for 2 hours. Similarly, after washing, 100 μl of HRP conjugated goat anti-mouse IgG (Sigma Aldrich) diluted 1:10,000 was added and reacted at room temperature for one hour. Finally, after the washing step, 100 μl of 3,3',5,5'-tetramethybenzidine (TMB) solution (BD bio sciences) was added to each well and reacted at room temperature for 30 minutes, after which 50 μl of _{2N H 2} SO _{4 was added.} After adding each well to stop the reaction, the OD ₄₅₀ value was measured through a microplate reader (FLUOstar Optima; BMG Labtech).

<6-3> Verification of neutralizing ability of antibody

에서 30분간 열불활성화(heat inactivation) 과정을 거친 후 2배 단계희석을 진행하여 최종 부피가 110μl가 되도록 하였다. 뎅기바이러스는 각 웰당 100μl를 접종하였을 때 50~60개의 플락이 형성될 수 있게 희석하여 최종 부피가 110μl가 되도록 한 후, 상기 혈청과 섞은 뒤 37

에서 90분간 반응시켰다. Vero cell이 배양되어있는 12well plate에 상기 혼합물을 웰당 200μl씩 접종한 다음 실온에서 한 시간 동안 배양한 후, 아가로즈-오버레이를 부어 굳힌 후 5% CO₂ 인큐베이터에서 37

로 4일간 배양하였다. NT titer는 항체를 섞지 않은 대조군과 비교하여 플락의 개수가 50% 줄어드는 시점을 기준으로 계산하였다.PRNT assay was performed to verify the ability of antibodies in serum obtained from blood samples to neutralize dengue virus. Serum after initial 1/20 dilution is 56

After the heat inactivation process was performed at for 30 minutes, a two-fold step dilution was performed so that the final volume became 110 μl. Dengue virus is diluted so that 50 to 60 flaks can be formed when 100 μl of each well is inoculated, the final volume is 110 μl, and then mixed with the serum 37

Reacted for 90 minutes. Inoculate 200 μl of the mixture per well into a 12 well plate in which Vero cells are cultured, incubate at room temperature for an hour, pour agarose-overlay, and harden, in a 5% CO ₂ incubator 37

And cultured for 4 days. The NT titer was calculated based on the time point at which the number of plaques decreased by 50% compared to the control group not mixed with the antibody.

<6-4> Check the result

As a result of measuring the antibody titer of Example <6-2>, the amount and reactivity of the produced antibody, that is, the antibody titer, was between'hRID-DV1ED3' not fused with CTB and'hRID-CTB-DV1ED3' fused with CTB. There was no significant difference (Fig. 8).

However, as a result of confirming the neutralizing ability against wild-type dengue virus through PRNT in Example <6-3>, it was confirmed that the neutralizing antibody titer showed a large difference depending on whether or not CTB was fused (FIG. 9). Specifically, FIG. 9 is a result of confirming the neutralizing ability for dengue virus serotype 1, and the neutralizing titer was measured based on the time point when the number of plaques became less than 50% compared to when only the virus was cultured without mixing serum. From this, when inoculated with hRID-CTB-DV1ED3, it was confirmed that the neutralization ability was increased to 200-300% compared to the case of inoculating only hRID-DV1ED3 without CTB fusion.

What is unique is that although the antibody titers measured by ELISA are almost the same, there is a large difference in neutralizing antibody titers. This actually means that the formation of pentamer through CTB fusion is very effective in the formation of antibodies directly involved in the neutralizing ability of the virus.

[Example 7]

Confirmation of water-soluble expression of various viral antigen proteins with icosahedral shell structure and formation of pentameric structure-Application to Japanese encephalitis virus

<7-1> Construction of recombinant vector and confirmation of water-soluble expression of recombinant protein

In order to verify that the technology of the present invention is applied to flaviviruses other than dengue virus, ED3 of Japanese encephalitis virus was expressed under the same conditions using the same vector system.

Specifically, in the same manner as in Example 1, the gene sequence encoding CTB (SEQ ID NO: 8) and the gene sequence encoding JEV ED3 (SEQ ID NO: 4) were inserted into the pGE-hRID plasmid using Kpn1 and Hind3 restriction enzymes. A recombinant vector in the form of -CTB-JEVED3 was prepared, and a vector was additionally prepared in the form of inserting Gly-Pro-Gly-Pro (GPGP) with a linker to create a flexible structure between CTB and JEVED3 (FIG. 10A). Each of the two recombinant vectors was transformed into SHuffle T7 cells to confirm the expression of the protein. In all subsequent experiments, there was no significant difference in the presence or absence of a GPGP linker.

As a result, it is possible to confirm the water-soluble expression efficiency of hRID-CTB-JEVED3 in [Fig. 10B], which is a result obtained by performing cell lysis in PBS conditions, and shows that the water-soluble efficiency is further increased under the A buffer condition used in actual purification. It can be seen in line S in Fig. 11.

<7-2> Purification of recombinant protein and confirmation of expression yield

The same nickel-affinity chromatography as in Example 2 was used to purify the recombinant protein in which the water-soluble expression was confirmed. Recombinant proteins were eluted according to a linear gradient from 10 mM to 300 mM imidazole. The eluted protein was subjected to SDS-PAGE for each fraction, and the fractions in which the recombinant protein was identified in FIG. (Up to fraction 23) were mixed and collected, concentrated and dialyzed for storage.

<7-3> Confirmation of formation of pentameric structure of recombinant protein

SDS-PAGE was performed to confirm whether the Japanese encephalitis virus recombinant protein obtained through the above purification had a pentameric structure. In the same manner as in Example 3, in order to maintain the structure of the protein in an undenaturation state, a loading buffer without DTT was used, and the boiling process was omitted.

As a result of comparing and measuring the band density, it can be seen that most of the recombinant proteins are formed as pentamers even though SDS is included (FIG. 12). As described in Example 3 above, when recalling that the physical properties of most proteins are mutually bonded to SDS and changed, this result suggests that the formed pentamer is a structurally highly stabilized standardized structure, which is the flavivirus technology of the present invention. It indicates that the antigen protein of can be expressed in a very stable pentameric form.

[Example 8]

Confirmation of water-soluble expression of various viral antigen proteins with icosahedral shell structure and formation of pentameric structure-application to foot-and-mouth disease virus

<8-1> Construction of recombinant vector and confirmation of water-soluble expression of recombinant protein

In addition, in order to confirm that the technology of the present invention is applicable to non-enveloped viruses, VP1 of the non-enveloped virus foot-and-mouth disease virus (FMDV) was expressed under the same conditions using a vector that expressed the recombinant protein of the enveloped virus. I did. In the same manner as in Example 1, a vector was prepared in the form of hRID-CTB-VP1 by inserting the gene sequence encoding CTB and FMDV VP1 using Kpn1 and Hind3 restriction enzymes in the pGE-hRID plasmid. To make, Gly-Pro-Gly-Pro (GPGP) was also produced in the form of inserting a linker. The control was prepared in the form of CTB-VP1 from which the hRID sequence was removed (Fig. 13A).

In Figure 13B, the amount of expression of the recombinant protein can be confirmed through SDS-PAGE, and the control without hRID (CTB-VP1) was not able to confirm the expression of the water-soluble protein, but the fusion protein (hRID-CTB) was fused with hRID. -VP1, hRID-CTB-GPGP-VP1) express 30-40% of the protein as water-soluble, which is a very improved result considering that the expression of the water-soluble protein hardly occurs in the control group.

<8-2> Purification of recombinant protein and confirmation of expression yield

The same nickel-affinity chromatography as in Example 2 was used to purify the recombinant protein in which the water-soluble expression was confirmed. Recombinant proteins were eluted according to a linear gradient from 10 mM to 300 mM imidazole. The eluted protein was subjected to SDS-PAGE for each fraction, and the fractions 21 to 25 were mixed and collected, concentrated and dialyzed to store the recombinant protein in FIG. 14.

<8-3> Confirmation of formation of pentameric structure of recombinant protein

SDS-PAGE was performed to confirm whether the non-enveloped viral recombinant protein obtained through the above purification had a pentameric structure. In the same manner as in Example 3, in order to maintain the structure of the protein in an undenaturation state, a loading buffer without DTT was used, and the boiling process was omitted. As a result of comparing and measuring the band density, it can be seen that most of the recombinant proteins are formed as pentamers even though SDS is included (FIG. 15). As described in Example 3 above, when recalling that the physical properties of most proteins are mutually bonded with SDS to change, this result suggests that the formed pentamer is a structurally highly stabilized standardized structure. This indicates that antigen proteins of both type virus and non-enveloped virus can be expressed in a very stable pentameric form.

[Example 9]

Confirmation of pentameric formation using E. coli heat-resistant enterotoxin B subunit

In the above experiments, it was confirmed that the pentamer of the fusion protein was formed using CTB, and in this example, it was replaced with E. coli heat-labile enterotoxin B subunit (LTB). A series of experiments were conducted in the same manner as the previous experiments, and the formation of a pentamer of the LTB fusion protein was confirmed in the same manner as in [Example 4] (FIG. 16). This result suggests that the pentameric toxin protein of the present invention is not limited to CTB, but can be flexibly applied to LTB or Shiga toxin belonging to the same family.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Pentamer-based recombinant protein vaccine platform and expressing system there of <130> 1064586 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 101 <212> PRT <213> Artificial Sequence <220> <223> Dengue virus ED3 <400> 1 Lys Gly Val Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys 1 5 10 15 Glu Val Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr 20 25 30 Glu Gly Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Ser Gln Asp Glu 35 40 45 Lys Gly Val Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val 50 55 60 Thr Asp Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly 65 70 75 80 Glu Ser Tyr Leu Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser 85 90 95 Trp Phe Lys Lys Gly 100 <210> 2 <211> 303 <212> DNA <213> Artificial Sequence <220> <223> Dengue virus ED3 <400> 2 aaaggcgtct cctacgtaat gtgtaccggc agcttcaagt tagaaaaaga ggttgccgag 60 acccagcatg ggaccgtact tgtccaggtg aagtacgagg gcactgatgc accctgtaaa 120 atccccttct ccagtcaaga cgagaagggg gtgacacaaa atggtcgtct gatcacagcg 180 aaccctatcg tgacggataa ggaaaagccc gttaacatcg aagcggaacc tccgtttggc 240 gaaagctatt tagtagtagg ggcgggggag aaagctctga agttatcgtg gttcaagaaa 300 ggc 303 <210> 3 <211> 104 <212> PRT <213> Artificial Sequence <220> <223> Japanese Encephalitis virus JEV <400> 3 Lys Gly Thr Thr Tyr Gly Met Cys Thr Glu Lys Phe Ser Phe Ala Lys 1 5 10 15 Asn Pro Ala Asp Thr Gly His Gly Thr Val Val Ile Glu Leu Ser Tyr 20 25 30 Ser Gly Ser Asp Gly Pro Cys Lys Ile Pro Ile Val Ser Val Ala Ser 35 40 45 Leu Asn Asp Met Thr Pro Val Gly Arg Leu Val Thr Val Asn Pro Phe 50 55 60 Val Ala Thr Ser Ser Ala Asn Ser Lys Val Leu Val Glu Met Glu Pro 65 70 75 80 Pro Phe Gly Asp Ser Tyr Ile Val Val Gly Arg Gly Asp Lys Gln Ile 85 90 95 Asn His His Trp His Lys Ala Gly 100 <210> 4 <211> 312 <212> DNA <213> Artificial Sequence <220> <223> Japanese Encephalitis virus JEV <400> 4 aaaggcacaa cctatggcat gtgcacagaa aaattctcgt tcgcgaaaaa tccggcggac 60 actggtcacg gaacagttgt cattgaactt tcctactctg ggagtgatgg cccttgcaaa 120 attccgattg tctccgttgc gagcctcaat gacatgaccc ccgtcgggcg gctggtgaca 180 gtgaacccct tcgtcgcgac ttccagcgcc aactcaaagg tgctagtcga gatggaaccc 240 cccttcggag actcctacat cgtagttgga aggggagaca agcagattaa ccaccattgg 300 cacaaggctg ga 312 <210> 5 <211> 211 <212> PRT <213> Artificial Sequence <220> <223> Foot-and-mouth disease virus VP1 <400> 5 Thr Thr Thr Thr Gly Glu Ser Ala Asp Pro Val Thr Thr Thr Val Glu 1 5 10 15 Asn Tyr Gly Gly Glu Thr Gln Thr Ala Arg Arg Leu His Thr Asp Val 20 25 30 Ala Phe Val Leu Asp Arg Phe Val Lys Leu Thr Gln Pro Lys Ser Thr 35 40 45 Gln Thr Leu Asp Leu Met Gln Ile Pro Ser His Thr Leu Val Gly Ala 50 55 60 Leu Leu Arg Ser Ala Thr Tyr Tyr Phe Ser Asp Leu Glu Val Ala Leu 65 70 75 80 Val His Thr Gly Pro Val Thr Trp Val Pro Asn Gly Ala Pro Lys Thr 85 90 95 Ala Leu Asn Asn His Thr Asn Pro Thr Ala Tyr Gln Lys Gln Pro Ile 100 105 110 Thr Arg Leu Ala Leu Pro Tyr Thr Ala Pro His Arg Val Leu Ser Thr 115 120 125 Val Tyr Asn Gly Lys Thr Thr Tyr Gly Glu Glu Ser Ser Arg Arg Gly 130 135 140 Asp Leu Ala Ala Leu Ala Arg Arg Val Asn Asn Arg Leu Pro Thr Ser 145 150 155 160 Phe Asn Tyr Gly Ala Val Lys Ala Asp Thr Ile Thr Glu Leu Leu Ile 165 170 175 Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro Arg Pro Leu Leu Ala Leu 180 185 190 Asp Thr Thr Gln Asp Arg Arg Lys Gln Lys Ile Ile Ala Pro Glu Lys 195 200 205 Gln Met Ile 210 <210> 6 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> Foot-and-mouth disease virus VP1 <400> 6 accactacca ccggtgaaag cgcagacccg gtaactacta ccgttgaaaa ctacgggggc 60 gaaacccaaa cggcgcgtcg tctgcatact gacgtagcgt tcgtgctgga ccgcttcgtt 120 aaactgaccc aaccgaaatc aactcagaca cttgatttaa tgcagatccc ttcccacacc 180 ctggtgggtg ctctattgcg atctgccacc tactacttca gcgatctgga agttgccctg 240 gtgcataccg ggccggttac ctgggttccg aacggcgctc cgaaaaccgc actcaacaac 300 cacaccaatc caacagcata tcagaaacag ccgatcaccc gtctggcgct gccttatact 360 gccccgcatc gtgttctgag cacggtgtac aacggtaaaa cgacctatgg cgaagaatcg 420 tctcgtcgtg gtgacctggc ggcgttggca cgtcgcgtga ataaccgcct gccgacctct 480 ttcaactacg gtgcggtaaa agccgatacc atcaccgaac tgctgatccg tatgaaacgt 540 gcggaaacct actgcccgcg cccgttgctg gcactggata ccacgcagga tcgccgtaaa 600 caaaaaatca tcgcaccgga aaaacagatg atc 633 <210> 7 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> cholera toxin B subunit (CTB) <400> 7 Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His Asn Thr Gln 1 5 10 15 Ile His Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu Ser Leu Ala 20 25 30 Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly Ala Thr Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu Thr Glu Ala 65 70 75 80 Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro His Ala Ile 85 90 95 Ala Ala Ile Ser Met Ala Asn 100 <210> 8 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> cholera toxin B subunit (CTB) <400> 8 actccgcaga acattacgga cctgtgtgcg gagtatcata atacgcagat tcacactttg 60 aatgacaaga ttttttcata tacggagtca ttagctggta aacgtgaaat ggcaattatc 120 acttttaaaa atggtgcgac gttccaggtg gaagttccgg gcagtcagca tattgatagt 180 cagaaaaaag ccatcgaacg tatgaaggat accttgcgta ttgcgtactt aaccgaggct 240 aaagtcgaga aattatgtgt ctggaataat aagaccccac atgccattgc tgcgatttcg 300 atggccaat 309 <210> 9 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> heat-labile enterotoxin B subunit (LTB) <400> 9 Ala Pro Gln Thr Ile Thr Glu Leu Cys Ser Glu Tyr Arg Asn Thr Gln 1 5 10 15 Ile Tyr Thr Ile Asn Asp Lys Ile Leu Ser Tyr Thr Glu Ser Met Ala 20 25 30 Gly Lys Arg Glu Met Val Ile Ile Thr Phe Lys Ser Gly Glu Thr Phe 35 40 45 Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln Lys Lys Ala 50 55 60 Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Thr Tyr Leu Thr Glu Thr 65 70 75 80 Lys Ile Asp Lys Leu Cys Val Trp Asn Asn Lys Thr Pro Asn Ser Ile 85 90 95 Ala Ala Ile Ser Met Lys Asn 100 <210> 10 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> heat-labile enterotoxin B subunit (LTB) <400> 10 gctccccaga ctattacaga actatgttcg gaatatcgca acacacaaat atatacgata 60 aatgacaaga tactatcata tacggaatcg atggcaggca aaagagaaat ggttatcatt 120 acatttaaga gcggcgaaac atttcaggtc gaagtcccgg gcagtcaaca tatagactcc 180 cagaaaaaag ccattgaaag gatgaaggac acattaagaa tcacatatct gaccgagacc 240 aaaattgata aattatgtgt atggaataat aaaaccccca attcaattgc ggcaatcagt 300 atgaaaaac 309 <210> 11 <211> 77 <212> PRT <213> Artificial Sequence <220> <223> human RNA interacting domain (hRID) <400> 11 Met Ser Glu Gln His Ala Gln Ala Ala Val Gln Ala Ala Glu Val Lys 1 5 10 15 Val Asp Gly Ser Glu Pro Lys Leu Ser Lys Asn Glu Leu Lys Arg Arg 20 25 30 Leu Lys Ala Glu Lys Lys Val Ala Glu Lys Glu Ala Lys Gln Lys Glu 35 40 45 Leu Ser Glu Lys Gln Leu Ser Gln Ala Thr Ala Ala Ala Thr Asn His 50 55 60 Thr Thr Asp Asn Gly Val Gly Pro Glu Glu Glu Ser Val 65 70 75 <210> 12 <211> 231 <212> DNA <213> Artificial Sequence <220> <223> human RNA interacting domain (hRID) <400> 12 atgtctgaac aacacgcaca ggcggccgtg caggcggccg aggtgaaagt ggatggcagc 60 gagccgaaac tgagcaagaa tgagctgaag agacgcctga aagctgagaa gaaagtagca 120 gagaaggagg ccaaacagaa agagctcagt gagaaacagc taagccaagc cactgctgct 180 gccaccaacc acaccactga taatggtgtg ggtcctgagg aagagagcgt g 231

Claims (16)

An N-terminal appended RNA interacting domain (RID) isolated from lysyl tRNA synthetase;
Pentameric toxin protein; And
A recombinant fusion protein expression vector comprising a polynucleotide encoding a protein of interest.
The method of claim 1,
The pentameric toxin protein is a cholera toxin B subunit (CTB) protein, an E. coli heat-labile enterotoxin B subunit (LTB) protein, and a Shiga toxin B subunit (Shiga-toxin). B subunit) will be selected from the group consisting of proteins.
The method of claim 1,
The target protein is an antigenic protein that induces an immune response and is a capsid protein of an icosahedral virus that forms a pentamer as a subunit in a self-assembly process.
The method of claim 1,
The target protein is a capsid protein of an icosahedral virus that forms a pentamer as a subunit in a self-assembly process, a vector.
The method of claim 4,
The icosahedral virus is an enveloped virus or a non-enveloped virus.
The method of claim 1,
The RID is represented by the amino acid sequence of SEQ ID NO: 11, vector.
The method of claim 2,
The CTB protein is represented by the amino acid sequence of SEQ ID NO: 7,
The LTB protein is represented by the amino acid sequence of SEQ ID NO: 9, the vector.
A host cell transformed with the expression vector according to claim 1.
The method of claim 8,
The host cell is a transformed host selected from the group consisting of bacteria of the genus Escherichia, bacteria of the genus Bacillus, bacteria of the genus Pseudomonas, lactic acid bacteria, yeast, animal cells, and insect cells. cell.
(a) N-terminal appended RNA interacting domain (RID) isolated from lysyl tRNA synthetase; Pentameric toxin protein; And preparing an expression vector comprising a polynucleotide encoding a protein of interest;
(b) preparing a transformant by introducing the expression vector into a host cell; And
(c) inducing the expression of the RID-CTB-target protein fusion protein by culturing the transformant; Method for producing a recombinant fusion protein comprising a.
Following paragraph 10,
The pentameric toxin protein is a cholera toxin B subunit (CTB) protein, an E. coli heat-labile enterotoxin B subunit (LTB) protein, and a Shiga toxin B subunit (Shiga-toxin). B subunit) is selected from the group consisting of proteins, production method.
The method of claim 10,
The host cells are bacteria of the genus Escherichia; Bacillus Bacillus bacteria; Bacteria of the genus Pseudomonas; Lactobacillus; leaven; Animal cells; And that is selected from the group consisting of insect cells, the production method.
An N-terminal appended RNA interacting domain (RID) isolated from lysyl tRNA synthetase; Cholera toxin B subunit (CTB) protein; And a protein of interest; a recombinant fusion protein comprising.
The method of claim 13,
The recombinant fusion protein is a recombinant fusion protein, characterized in that in the form of a pentamer.
A composition for diagnosing viral infection, comprising the expression vector according to claim 1 or the recombinant fusion protein according to claim 13.
A vaccine composition for enhancing immunity comprising the expression vector according to claim 1 or the recombinant fusion protein according to claim 13.

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