Pentameric toxin protein-based target protein fusion pentamer production platform using viral nucleocapsids

Abstract

The expression vector for producing the target protein-fused pentamer of the present invention contains polynucleotides encoding a pentameric toxin protein and a viral nucleocapsid as fusion partners of the target protein. Further, the method for producing the target protein-fused pentamer of the present invention includes the steps of: producing an expression vector for producing the target protein-fused pentamer, which contains a target protein and polynucleotides encoding a pentameric toxin protein and a viral nucleocapsid as fusion partners of the target protein; introducing the expression vector for producing the target protein-fused pentamer into a host cell to produce a transformant; and culturing the transformant.

Description

Technical Field

[0001] The present invention relates to a production platform for a target protein fusion pentamer based on a pentameric toxin protein using a viral nucleocapsid. More specifically, the present invention relates to an expression vector capable of efficiently producing a target protein in the form of a pentamer in a host cell using a pentameric toxin protein and a viral nucleocapsid as fusion partners, a host cell transformed with the expression vector, and a method for producing a target protein fusion pentamer using the same.

Background Art

[0002] There are various systems for producing proteins, but from the viewpoint of mass, rapid, and inexpensive production, it is preferable to use a prokaryotic cell system such as Escherichia coli if possible. However, in such a system, there is a problem that many proteins, particularly eukaryotic cell-derived proteins and viral proteins that infect them, lose their water solubility and are produced as insoluble precipitates.

[0003] In addition, for the merit of increasing vaccine efficacy and the like, techniques for producing antigen proteins in the form of multimers have been studied, and as one of such techniques, a method of utilizing a toxin protein that forms a pentameric structure has been attempted. Toxin proteins belonging to the heat-labile enterotoxin (LT) family are known to form such pentameric structures, and in the case of cholera toxin B subunit (CTB), which belongs to this family, it is usefully used as a carrier function that promotes the mucosal absorption of chemically or genetically conjugated foreign antigens.

[0004] There have been attempts to produce such pentameric toxin protein-based fusion proteins in prokaryotic cell systems such as Escherichia coli for large-scale, rapid, and inexpensive production. However, due to protein folding-related problems and the like, they are produced as insoluble precipitates, and there is also a problem that it is difficult to produce the desired pentamer even after undergoing processes such as refolding. Therefore, it is necessary to develop a technology to solve this problem.

Summary of the Invention

Problems to be Solved by the Invention

[0005] The main object of the present invention is to provide an expression vector capable of efficiently producing a target protein, particularly a difficult-to-express protein that is insoluble and expressed in Escherichia coli, in the form of a pentamer in a host cell, particularly Escherichia coli.

[0006] Another object of the present invention is to provide a host cell transformed with the above expression vector and capable of realizing efficient production of the above pentamer.

[0007] Still another object of the present invention is to provide a method capable of efficiently producing the above pentamer using the above expression vector and / or the above host cell.

Means for Solving the Problems

[0008] The present invention provides an expression vector for producing a target protein fusion pentamer, which contains a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a fusion partner of the target protein.

[0009] The present invention also provides a host cell transformed with the expression vector for producing the target protein fusion pentamer.

[0010] The present invention also provides a method for producing a target protein fusion pentamer, comprising the steps of: preparing an expression vector for producing a target protein fusion pentamer, which contains a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a target protein and a fusion partner of the target protein; introducing the expression vector for producing the target protein fusion pentamer into a host cell to produce a transformant; and culturing the transformant.

Advantages of the Invention

[0011] According to the present invention, a target protein, particularly a difficult-to-express protein that is insoluble when expressed in Escherichia coli, can be efficiently produced in a host cell, particularly Escherichia coli, in the form of a pentamer. In particular, according to the present invention, a vaccine antigen with greatly improved immunogenicity can be efficiently produced by the formation of a sophisticated pentamer.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0013] According to one embodiment of the present invention, there is provided an expression vector for producing a target protein fusion pentamer comprising a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a fusion partner of the target protein.

[0014] As used herein, the term "target protein" means any protein that a person skilled in the art intends to produce in large quantities, and means all proteins that can be expressed in a host cell by inserting a polynucleotide encoding the protein into a recombinant vector.

[0015] In the present invention, the target protein can also be selected from one or more of the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum, and a cell protein, but is not limited thereto.

[0016] In the present invention, the target protein can be a viral antigen protein that induces an immune response. For example, it can be a virus having a spherical icosahedral capsid structure, wherein the icosahedron is a structure formed by gathering 20 equilateral triangles as small units, and the 12 vertices have large two-fold axes, three-fold axes, and five-fold axes, and the small units consist of a combination of pentagons and hexagons according to the axes. It can be an antigen protein of a virus.

[0017] In the present invention, the target protein can be the capsid protein of an icosahedral virus that forms a pentamer (or hexamer) as a small unit in the self-assembly process. When such a protein is used as the target protein, it is possible to produce a recombinant pentamer having the same level of antigenicity as the pentamer of the original viral capsid protein, particularly in E. coli.

[0018] For this, pentameric structural antigens of all enveloped or non-enveloped viruses that generally constitute an icosahedron can be applied.

[0019] Examples of enveloped viruses having a lipid membrane may include viruses of the genus Herpesviridae and Hepadnaviridae having a DNA genotype, and viruses of the genus Flaviviridae having an RNA genotype.

[0020] Examples of non-enveloped viruses (non-enveloped or naked viruses) composed of viral envelope proteins without a lipid membrane may include viruses of the genus Adenoviridae and Parvoviridae having a DNA genotype, and viruses of the genus Reoviridae, Picornaviridae, and Caliciviridae having an RNA genotype.

[0021] As used herein, the term "expression vector" is a vector for introducing and expressing a polynucleotide encoding a polypeptide of interest into a heterologous or homologous host cell, and means a circular or linear polynucleotide, for example, a DNA or RNA molecule. The expression vector may contain a promoter and / or a terminator sequence, or may also contain components necessary for use in vectors such as an origin of replication, a selection marker, a polyadenylation signal, etc.

[0022] The expression vector of the present invention can be a plasmid, a viral vector, a phage particle or a genomic insert, and after being introduced into a host cell, it can be replicated regardless of the genome of the host cell or integrated into the genome of the host cell.

[0023] The expression vector of the present invention may contain a polynucleotide encoding a target protein, or may be for use including a polynucleotide encoding a future target protein. Particularly in the latter case, preferably, as a component that can easily include a polynucleotide encoding a target protein, it may usually contain a restriction enzyme recognition site called a multi-cloning site (MCS).

[0024] When the present invention applies a pentameric toxin protein and a viral nucleocapsid as fusion partners of a target protein, it can efficiently express a protein that is insoluble when not using a fusion partner or using other conventional fusion partners as a water-soluble protein. Based on new research results that such a water-soluble expressed target protein can efficiently produce self-assembled pentamers.

[0025] When using such a fusion partner, there is an advantage that the target protein can be produced with enhanced water solubility. Generally, the larger the size of the fusion partner, the more useful it is for enhancing the water solubility of the target protein. However, depending on the size of the fusion partner, the structural interference of the immediately adjacent target protein can deepen, and it can also interfere with the production of the physiological active structure. Therefore, if possible, it is very important to discover a fusion partner that enhances the water solubility and activity of the target protein despite its small size.

[0026] In particular, the viral nucleocapsid used as a fusion partner in the present invention has the advantage that, because of its very small size (short peptide length), it can minimize structural interference not only with the target protein but also with the self-assembly of target protein monomers (in the case of the present invention, self-assembly into pentamers).

[0027] Moreover, according to the present invention, there is an advantage that the zinc binding domain inherent to the viral nucleocapsid can be used. For example, when using the zinc binding domain of the viral nucleocapsid, for example, when using a zinc affinity purification method, the fusion protein can be easily purified even without a separate component for purification such as a histidine tag. In the present invention, the viral nucleocapsid may be a conventionally known viral nucleocapsid, as long as the activity of the present invention, that is, the activity of acting as a fusion partner for the target protein together with the pentameric toxin protein, enabling the target protein to be expressed in a water-soluble manner, particularly in Escherichia coli, while maintaining the ability to form pentamers, it may be a fragment or mutant of the viral nucleocapsid.

[0028] In the present invention, the viral nucleocapsid can be a nucleocapsid of a retrovirus, such as a retrovirus such as HIV-1, SIV, MuLV, etc.

[0029] In the present invention, the viral nucleocapsid is preferably a nucleocapsid of the human immunodeficiency virus (HIV).

[0030] In the present invention, the viral nucleocapsid preferably has an amino acid sequence having a sequence identity of 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, yet more preferably 90% or more, still more preferably 95% or more, yet more preferably 96% or more, still more preferably 97% or more, yet more preferably 98% or more, still more preferably 99% or more with the amino acid sequence of SEQ ID NO: 1, or includes the amino acid sequence of SEQ ID NO: 1. Preferably, while maintaining the sequences at positions 14, 17, 22 and 27 in SEQ ID NO: 1, more preferably while maintaining the sequences at positions 14, 17, 22, 27, 35, 38, 43 and 48, still more preferably while maintaining the sequences at positions 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48 and 51, it includes an amino acid sequence satisfying the above sequence identity.

[0031] In the present invention, the polynucleotide encoding the viral nucleocapsid preferably has a base sequence having a sequence identity of 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, yet more preferably 90% or more, still more preferably 95% or more, yet more preferably 96% or more, still more preferably 97% or more, yet more preferably 98% or more, still more preferably 99% or more with the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3, or includes the base sequence of SEQ ID NO: 2 or SEQ ID NO: 3. Preferably, while maintaining the sequences at positions 14, 17, 22 and 27 in SEQ ID NO: 1, more preferably while maintaining the sequences at positions 14, 17, 22, 27, 35, 38, 43 and 48, still more preferably while maintaining the sequences at positions 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48 and 51, it includes a base sequence satisfying the above sequence identity while encoding an amino acid sequence.

[0032] As used herein, sequence identity means the sequence identity between an amino acid sequence and a reference amino acid sequence or between a nucleotide sequence and a reference nucleotide sequence. Sequence identity can be determined by comparing the positions of each sequence that can be aligned for comparison purposes. When the positions of the compared sequences are occupied by the same amino acid or base, the molecules are identical at that position. The degree of identity between amino acid or nucleotide sequences is a function of the number of identical amino acids or nucleotides at positions shared by the respective amino acid or nucleotide sequences. For example, the identity between two sequences can be calculated using various alignment algorithms and / or programs including FASTA or BLAST.

[0033] In the present invention, the pentameric toxin protein is a toxin protein having an activity such that each can form a complex protein in the pentamer form as a unit, and may be a fragment of the pentameric toxin protein as long as the activity of forming a complex protein in the pentamer form is maintained, including, of course, conventionally known pentameric toxin proteins. For example, it may also be selected from the group consisting of cholera toxin B subunit (CTB), heat-labile enterotoxin B subunit (LTB) of Escherichia coli, and Shiga-toxin B subunit.

[0034] In the present invention, the pentameric toxin protein preferably has a sequence identity of 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, more preferably 98% or more, still more preferably 99% or more with the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6, or includes the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 6.

[0035] In the present invention, the polynucleotide encoding the pentameric toxin protein preferably has a nucleotide sequence having a sequence identity of 50% or more, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, more preferably 96% or more, still more preferably 97% or more, more preferably 98% or more, still more preferably 99% or more with the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or contains the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 7.

[0036] The expression vector of the present invention may be for producing a target protein fusion pentamer in cells selected from all organisms including prokaryotes and eukaryotes, but is preferably for producing a target protein fusion pentamer in a microorganism of the genus Escherichia, and more preferably for producing a target protein fusion pentamer in Escherichia coli.

[0037] In the expression vector of the present invention, the polynucleotides encoding the pentameric toxin protein and the viral nucleocapsid may further contain other components, for example, a cloning site for inserting a polynucleotide encoding a target protein, for example, a restriction enzyme recognition site, for example, a multiple cloning site (MCS); a linker sequence for imparting a spacer between the target protein and the fusion partner and / or between the fusion partners (between the pentameric toxin protein and the viral nucleocapsid); a tag sequence, for example, a histidine tag sequence; and / or a protease recognition site coding sequence for excising a part of the expressed fusion protein. Each of these may be arranged in various ways as necessary and may be included in various numbers.

[0038] In one embodiment, the expression vector of the present invention includes a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a fusion partner of a target protein, and the polynucleotide encodes a pentameric toxin protein and a viral nucleocapsid bound to the N-terminus of the pentameric toxin protein, and includes a cloning site for binding a target protein or a target protein to the C-terminus of the pentameric toxin protein. As a specific exemplification of such an embodiment, the polynucleotide includes a construct in which a promoter, a viral nucleocapsid coding site, a pentameric toxin protein coding site, a target protein coding site or a cloning site (for example, a restriction enzyme recognition site, for example, a multiple cloning site (MCS)) for inserting a target protein coding polynucleotide, and a terminator are operably linked in the 5' to 3' direction.

[0039] In another embodiment, the expression vector of the present invention includes a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a fusion partner of a target protein, and the polynucleotide encodes a pentameric toxin protein and a viral nucleocapsid bound to the C-terminus of the pentameric toxin protein, and includes a cloning site for binding a target protein or a target protein between the pentameric toxin protein and the viral nucleocapsid. As a specific exemplification of such an embodiment, the polynucleotide includes a construct in which a promoter, a pentameric toxin protein coding site, a target protein coding site or a cloning site (for example, a restriction enzyme recognition site, for example, a multiple cloning site (MCS)) for inserting a target protein coding polynucleotide, a viral nucleocapsid coding site, and a terminator are operably linked in the 5' to 3' direction.

[0040] The expression vector of the present invention may contain a T7 promoter and a T7 terminator such that the transcription of the target protein and the fusion partner-encoding DNA is regulated by the bacteriophage T7 system. According to this, more effective production of the target protein in Escherichia coli is possible.

[0041] The expression vector of the present invention as described above can be produced, for example, using an ordinary recombination method based on a conventional expression vector.

[0042] According to another embodiment of the present invention, there is provided a host cell transformed with an expression vector for producing the target protein fusion pentamer of the present invention.

[0043] In the present invention, the host cell may be selected from cells of all organisms including prokaryotes and eukaryotes, but preferably is a cell of a microorganism of the genus Escherichia, and more preferably Escherichia coli.

[0044] As used herein, the terms "transformation" or "introduction" mean introducing a polynucleotide into a host such that the polynucleotide becomes replicable as an extrachromosomal factor or by completion of chromosomal integration. Methods for transforming with the expression vector according to the present invention may include, but are not limited to, electroporation, calcium phosphate (CaPO4) method, calcium chloride (CaCl2) method, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method or lithium acetate-DMSO method.

[0045] According to still another embodiment of the present invention, there is provided a method for producing a target protein fusion pentamer, comprising the steps of: preparing an expression vector for producing a target protein fusion pentamer, the expression vector containing a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a target protein and a fusion partner of the target protein; introducing the expression vector for producing the target protein fusion pentamer into a host cell to produce a transformant; and culturing the transformant.

[0046] At this time, for specific matters such as matters related to the expression vector and matters related to the introduction of the expression vector, reference may be made to the matters described above.

[0047] For culturing the transformant, a culturing method known to be suitable for the host cell can be used. For example, when the host cell is Escherichia coli, a method of culturing in an LB (Luria - Bertani) medium at a temperature of 15 to 39°C can be used. If the expression vector has a component of an antibiotic selection marker (for example, an ampicillin resistance gene of an antibiotic), at this time, by adding the antibiotic (for example, ampicillin) to the medium and culturing, the growth of Escherichia coli containing the expression vector can be preferentially induced. Further, if the expression vector has a component for regulating the transcription and / or translation of the target protein - encoding DNA, for example, a lac - operon - related component, a related substance, for example, IPTG, can be added to induce the expression of the target protein.

[0048] The following examples are presented to assist in understanding the invention, and the following content does not limit the protection scope of the invention.

Example

[0049] [Example] Production Example 1 To investigate the feasibility of using a combination of a pentameric toxin protein and a viral nucleocapsid (NC) as fusion partners for the production of pentamers, a vector was constructed for expressing a form in which the NC of human immunodeficiency virus (HIV) was linked to the N-terminus of the pentameric toxin protein, heat-labile enterotoxin B subunit (LTB) of Escherichia coli, and six histidines were linked to the C-terminus of LTB (Construct 1 in Figure 1).

[0050] At this time, a vector was constructed based on the pGEMEX-1 (Promega) vector designed such that the expression of the target protein is regulated by the T7 promoter. The sequence of SEQ ID NO: 5 was used as the sequence encoding LTB, and the sequence of SEQ ID NO: 2 (or SEQ ID NO: 3) was used as the sequence encoding NC.

[0051] In the constructed vector, the sequence of the construct site is the same as SEQ ID NO: 8, and the amino acid sequence of the expected expressed fusion protein is the same as SEQ ID NO: 9.

[0052] Example 1 A vector was constructed for producing a pentamer of a fusion protein in which NC was linked to the N-terminus of LTB, a pentameric toxin protein, a Tobacco Etch Virus (TEV) protease cleavage site and six histidines were present between them, and VP1 of Poliovirus, the target protein, was linked to the C-terminus of LTB (Construct 2 in Figure 1).

[0053] It was constructed based on the pGEMEX-1 (promega) vector in the same manner as in Production Example 1.

[0054] In the constructed vector, the sequence of the construct site is the same as SEQ ID NO: 10, and the amino acid sequence of the expected expressed fusion protein is the same as SEQ ID NO: 11.

[0055] Example 2 NC was linked to the N-terminus of cholera toxin B subunit (CTB) of Vibrio cholerae, which is a pentameric toxin protein, and a TEV protease cleavage site and six histidines were present between them. A vector was constructed to produce a pentamer of a fusion protein in which VP1 of poliovirus, the target protein, was linked to the C-terminus of CTB (construct 3 in Figure 1).

[0056] It was constructed based on the pGEMEX-1 (Promega) vector in the same manner as in Production Example 1.

[0057] In the constructed vector, the sequence of the construct site is the same as SEQ ID NO: 12, and the amino acid sequence of the expected expressed fusion protein is the same as SEQ ID NO: 13.

[0058] Comparative Example 1 EPRS was linked to the N-terminus of LTB, which is a pentameric toxin protein, instead of NC, and a TEV protease cleavage site and six histidines were present between them. A vector was constructed to produce a pentamer of a fusion protein in which VP1 of poliovirus, the target protein, was linked to the C-terminus of LTB (construct 4 in Figure 1).

[0059] Here, EPRS is the WHEP domain of human-derived Glutamyl-prolyl-tRNA synthetase (the WHEP domain located in the middle of EPRS, including TRS-1, TRS-2, TRS-3, and the linker connecting the three domains).

[0060] It was constructed based on the pGEMEX-1 (Promega) vector in the same manner as in Production Example 1.

[0061] Example 3 NC was linked to the N-terminus of LTB, which is a pentameric toxin protein, and six histidines and a TEV protease cleavage site were present between them. A vector was constructed to produce a pentamer of a fusion protein in which VP1 of foot-and-mouth disease virus (FMDV), the target protein, was linked to the C-terminus of LTB (construct 5 in Figure 1).

[0062] Based on the pGEMEX-1 (Promega) vector, it was constructed in the same manner as in Production Example 1.

[0063] In the constructed vector, the sequence of the construct site is the same as SEQ ID NO: 14, and the amino acid sequence of the expected expressed fusion protein is the same as SEQ ID NO: 15.

[0064] Example 4 VP1 of foot-and-mouth disease virus (FMDV), the target protein, was linked to the C-terminus of LTB, which is a pentameric toxin protein. NC was linked to the C-terminus of FMDV VP1, and six histidines were linked to the C-terminus of NC. A vector was constructed to produce a pentamer of a fusion protein in this form (construct 6 in Figure 1).

[0065] Based on the pGEMEX-1 (promega) vector, it was constructed in the same manner as in Production Example 1.

[0066] In the constructed vector, the sequence of the construct site is the same as SEQ ID NO: 16, and the amino acid sequence of the expected expressed fusion protein is the same as SEQ ID NO: 17.

[0067] Comparative Example 2 EPRS was linked to the N-terminus of LTB, which is a pentameric toxin protein, instead of NC, and six histidines and a TEV protease cleavage site were present between them. A vector was constructed to produce a pentamer of a fusion protein in which VP1 of foot-and-mouth disease virus, the target protein, was linked to the C-terminus of LTB (construct 7 in Figure 1).

[0068] Based on the pGEMEX-1 (Promega) vector, it was prepared in the same manner as in Production Example 1.

[0069] Experimental Example The vectors of the above Production Examples, Examples, and Comparative Examples were transformed into Escherichia coli Shuffle / T7 / pLysS and cultured. Shuffle / T7 / pLysS is BL21 star(DE3)pLysS One Shot TM (Invitrogen TM )competent Escherichia coli. The pLysS plasmid was extracted through Mini-prep, introduced into SHuffle T7 (NEB) competent Escherichia coli, and then selected with chloramphenicol (CM), an antibiotic marker contained in pLysS. All transformed Escherichia coli were cultured in LB medium containing 50 μg / ml of ampicillin and 34 μg / ml of chloramphenicol. The culture temperature was set at 16 - 37°C. When the OD600 value of Escherichia coli reached 0.5 or more, IPTG was added at a level of 0 μM - 1 mM to activate the T7 promoter, and after adding IPTG, it was cultured at 30°C for 3 hours or at 16 - 20°C for about 16 hours so that proteins could be produced sufficiently. The sufficiently cultured Escherichia coli were centrifuged, the supernatant was removed, and then stored. Next, 0.3 ml of PBS was added to the Escherichia coli harvest corresponding to 5 ml of LB medium, and ultrasonication was performed to prepare a lysate. Then, the lysate was centrifuged and separated into a soluble fraction and a pellet fraction, and the total lysate, soluble fraction, and pellet fraction were classified and analyzed by SDS-PAGE.

[0070] First, the vector of Production Example 1 was introduced into Escherichia coli to induce the expression of the fusion protein. As a result, as shown in Fig. 2, it was shown that the protein was expressed normally, that is, in the expected size and water-soluble form, and in a large amount. This indicates that the combination of the pentameric toxin protein and NC may be utilized as a fusion partner for the production of the target protein-fused pentamer.

[0071] The vector of Example 1 was introduced into Escherichia coli to induce the expression of the fusion protein. As a result, as shown in Fig. 3, it was shown that the protein was expressed normally. In particular, LTB is known to be expressed in an insoluble form in Escherichia coli and it is very difficult to express it normally. Therefore, such results indicate that the method of the present invention can express a protein that is conventionally expressed in an insoluble form in Escherichia coli in a water-soluble form.

[0072] The vector of Example 2 was introduced into Escherichia coli to induce the expression of the fusion protein. As a result, as shown in Fig. 4, it was also shown that the protein was expressed normally.

[0073] The vector of Example 3 was introduced into Escherichia coli to induce the expression of the fusion protein. As a result, as shown in Fig. 5, it was also shown that the protein was expressed normally.

[0074] The vector of Example 4 was introduced into Escherichia coli to induce the expression of the fusion protein. As a result, as shown in Fig. 6, it was also shown that the protein was expressed normally. In this case, since the arrangement of the pentameric toxin protein, the target protein, and NC was changed, such results indicate that normal expression is possible even when the arrangement of each component is changed.

[0075] In addition, the products obtained through the induction of expression of each vector in the above Production Examples, Examples, and Comparative Examples, that is, the pentameric GM1-binding ability, was investigated. Toxin proteins belonging to the enterotoxin family form pentamers on the surface of the intestinal mucosa and exhibit the ability to bind to the GM1 ganglioside receptor. Therefore, the ability of the expression product to bind to GM1 ganglioside means that pentamers are well formed, indicating that the protein is not simply expressed in a water-soluble form but has a correctly formed structure. Thus, an attempt was made to confirm this through the investigation of GM1-binding ability.

[0076] Specifically, GM1 was diluted in bicarbonate buffer (pH 9.6), dispensed into immunoplates (Maxisorp, Thermo Scientific), and coated at 4°C for approximately 16 hours. Next, it was washed three times with PBS-T (0.1% Tween-20). After preparing the expression product samples by diluting them two-fold (serial dilution with the initial dilution concentration adjusted to 100 nM), they were placed in each well and reacted at room temperature for 4 hours to ensure sufficient binding to GM1. Then, as the primary antibody, anti-6His tag was diluted 1:2,000 in serum dilution buffer (PBS-T with 0.25% BSA) and placed in each well of the plate, and reacted at room temperature for 1 hour. Subsequently, as the secondary antibody, anti-mouse (HRP conjugated) was diluted 1:10,000 in serum dilution buffer, placed in each well of the plate, and reacted at room temperature for 1 hour. After reacting each of the primary and secondary antibodies, it was washed with PBS-T. After the reaction of the secondary antibody, TMB (BD OptEIA TMB Substrate Reagent Set) was placed in each well to induce the color development of HRP. After 30 minutes, 2N sulfuric acid was added to terminate the reaction, and then it was analyzed using an ELISA reader.

[0077] As a result of investigating the GM1-binding ability of Production Example 1, Example 1, Example 2, and Comparative Example 1, as shown in Figure 7, it was shown that the GM1-binding ability of the Examples was superior to that of Production Example 1 and Comparative Example 1.

[0078] As a result of investigating the GM1 binding ability of Production Example 1, Example 3, Example 4, and Comparative Example 2, as shown in Fig. 8, it was shown that the GM1 binding ability of the examples was excellent compared to Production Example 1 and Comparative Example 2.

[0079] As described above, the present invention has been described mainly with reference to its preferred embodiments. Those having ordinary knowledge in the technical field to which the present invention pertains can understand that the present invention can be embodied in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting perspective. The scope of the present invention is shown not in the foregoing description but in the claims, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

[0080] <Sequence Listing> SEQ ID NO:1 Sequence Name: Amino Acid Sequence of HIV Nucleocapsid Sequence Type: AA Organism Name: Human Immunodeficiency Virus Sequence: QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQAN

[0081] SEQ ID NO:2 Sequence Name: Nucleotide Sequence Encoding HIV Nucleocapsid Sequence Type: DNA Organism Name: Human Immunodeficiency Virus Sequence: CAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAAC

[0082] SEQ ID NO:3 Array Name: Nucleotide Sequence Encoding HIV Nucleocapsid Array Type: DNA Organism Name: Synthetic Construct Sequence: CAGCGTGGTAACTTCCGTAACCAGCGTAAAACCGTTAAATGCTTCAACTGCGGCAAAGAAGGCCACATCGCGAAAAACTGCCGTGCGCCGCGTAAAAAAGGCTGCTGGCGTTGCGGCCGTGAAGGCCACCAGATGAAAGATTGCACCGAACGTCAGGCGAAC

[0083] SEQ ID NO:4 Array Name: Amino Acid Sequence of Heat-Labile Enterotoxin B Subunit Derived from Escherichia coli Array Type: AA Organism Name: Escherichia coli Sequence: APQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKN

[0084] SEQ ID NO:5 Array Name: Nucleotide Sequence Encoding Heat-Labile Enterotoxin B Subunit Derived from Escherichia coli Array Type: DNA Organism Name: Escherichia coli Sequence: GCTCCCCAGACTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGAAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAGAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGAAAAAC

[0085] SEQ ID NO:6 Sequence Name: Amino Acid Sequence of Cholera Toxin B Subunit Derived from Vibrio cholerae Sequence Type: AA Organism Name: Vibrio cholerae Sequence: TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN

[0086] SEQ ID NO:7 Sequence Name: Nucleotide Sequence Encoding Cholera Toxin B Derived from Vibrio cholerae Sequence Type: DNA Organism Name: Vibrio cholerae Sequence: ACTCCGCAGAACATTACGGACCTGTGTGCGGAGTATCATAATACGCAGATTCACACTTTGAATGACAAGATTTTTTCATATACGGAGTCATTAGCTGGTAAACGTGAAATGGCAATTATCACTTTTAAAAATGGTGCGACGTTCCAGGTGGAAGTTCCGGGCAGTCAGCATATTGATAGTCAGAAAAAAGCCATCGAACGTATGAAGGATACCTTGCGTATTGCGTACTTAACCGAGGCTAAAGTCGAGAAATTATGTGTCTGGAATAATAAGACCCCACATGCCATTGCTGCGATTTCGATGGCCAAT

[0087] SEQ ID NO:8 Sequence name: Nucleotide sequence of the fusion protein construct Sequence type: DNA Organism name: Synthetic construct Sequence: ATGCAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAACGGATCCGCTCCCCAGACTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGAAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAGAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGAAAAAC

[0088] SEQ ID NO:9 Array Name: Amino acid sequence of the expected fusion protein Array Type: AA Organism Name: Synthetic construct Sequence: MQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANGSAPQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKN

[0089] SEQ ID NO:10 Array Name: Nucleotide sequence of the fusion protein construct Array Type: DNA Organism Name: Synthetic construct Sequence:

[0090] SEQ ID NO:11 Array Name: Amino acid sequence of the expected fusion protein Array Type: AA Organism Name: Synthetic construct Sequence: QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDGTGSAPQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNDIGLGQMLESMIDNTVRETVGAATSRDALPNTEASGPTHSKEIPALTAVETGATNPLVPSDTVQTRHVVQHRSRSESSIESFFARGACVTIMTVDNPASTTNKDKLFAVWKITYKDTVQLRRKLEFFTYSRFDMELTFVVTANFTETNNGHALNQVYQIMYVPPGAPVPEKWDDYTWQTSSNPSIFYTYGTAPARISVPYVGISNAYSHFYDGFSKVPLKDQSAALGDSLYGAASLNDFGILAVRVVNDHNPTKVTSKIRVYLKPKHIRVWCPRPPRAVAYYGPGVDYKDGTLTPLSTKDLTTY

[0091] SEQ ID NO:12 Array Name: Nucleotide sequence of the fusion protein construct Array Type: DNA Organism Name: Synthetic construct Sequence:

[0092] SEQ ID NO:13 Array Name: Amino acid sequence of the expected fusion protein Array Type: AA Organism Name: Synthetic construct Sequence: QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDGTGSTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANDIGLGQMLESMIDNTVRETVGAATSRDALPNTEASGPTHSKEIPALTAVETGATNPLVPSDTVQTRHVVQHRSRSESSIESFFARGACVTIMTVDNPASTTNKDKLFAVWKITYKDTVQLRRKLEFFTYSRFDMELTFVVTANFTETNNGHALNQVYQIMYVPPGAPVPEKWDDYTWQTSSNPSIFYTYGTAPARISVPYVGISNAYSHFYDGFSKVPLKDQSAALGDSLYGAASLNDFGILAVRVVNDHNPTKVTSKIRVYLKPKHIRVWCPRPPRAVAYYGPGVDYKDGTLTPLSTKDLTTY

[0093] SEQ ID NO:14 Array Name: Nucleotide sequence of the fusion protein construct Array Type: DNA Organism Name: Synthetic construct Sequence:

[0094] SEQ ID NO:15 Array Name: Amino Acid Sequence of the Expected Fusion Protein Array Type: AA Organism Name: Synthetic Construct Sequence: QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDGTGSAPQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNDITTTTGESADPVTTTVENYGGETQTARRLHTDVAFVLDRFVKLTQPKSTQTLDLMQIPSHTLVGALLRSATYYFSDLEVALVHTGPVTWVPNGAPKTALNNHTNPTAYQKQPITRLALPYTAPHRVLSTVYNGKTTYGEESSRRGDLAALARRVNNRLPTSFNYGAVKADTITELLIRMKRAETYCPRPLLALDTTQDRRKQKIIAPEKQMI

[0095] SEQ ID NO:16 Array Name: Nucleotide Sequence of the Fusion Protein Construct Array Type: DNA Organism Name: Synthetic Construct Sequence:

[0096] SEQ ID NO:17 Array Name: Amino acid sequence of the expected fusion protein Array Type: AA Organism Name: Synthetic construct Array: APQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNGSTTTTGESADPVTTTVENYGGETQTARRLHTDVAFVLDRFVKLTQPKSTQTLDLMQIPSHTLVGALLRSATYYFSDLEVALVHTGPVTWVPNGAPKTALNNHTNPTAYQKQPITRLALPYTAPHRVLSTVYNGKTTYGEESSRRGDLAALARRVNNRLPTSFNYGAVKADTITELLIRMKRAETYCPRPLLALDTTQDRRKQKIIAPEKQMIDIQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANKLHHHHHH

Claims

1. An expression vector for producing a target protein fusion pentamer, comprising a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a fusion partner of the target protein.

2. The expression vector for producing a target protein fusion pentamer according to claim 1, wherein the pentameric toxin protein is selected from the group consisting of cholera toxin B subunit (CTB), heat-labile enterotoxin B subunit (LTB) of Escherichia coli, and Shiga-toxin B subunit.

3. The expression vector for producing a target protein fusion pentamer according to claim 1, wherein the target protein is a capsid protein of an icosahedral virus that forms a pentamer in a small unit during the self-assembly process.

4. The expression vector for producing a target protein fusion pentamer according to claim 1, wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus.

5. The expression vector for producing a target protein fusion pentamer according to claim 1, wherein the viral nucleocapsid contains the amino acid sequence of SEQ ID NO:

1.

6. The expression vector for producing a target protein fusion pentamer according to claim 1, wherein the vector is for producing a target protein fusion pentamer in Escherichia coli.

7. A host cell transformed with the expression vector for producing a target protein fusion pentamer according to any one of claims 1 to 6.

8. Producing an expression vector for producing a target protein fusion pentamer, comprising a target protein and a polynucleotide encoding a pentameric toxin protein and a viral nucleocapsid as a fusion partner of the target protein; introducing the expression vector for producing a target protein fusion pentamer into a host cell to produce a transformant; and culturing the transformant; A method for producing a target protein fusion pentamer comprising the steps of:

9. The method for producing a target protein fusion pentamer according to claim 8, wherein the pentameric toxin protein is selected from the group consisting of cholera toxin B subunit (CTB), heat-labile enterotoxin B subunit (LTB) of Escherichia coli, and Shiga-toxin B subunit.

10. The method for producing a target protein fusion pentamer according to claim 8, wherein the target protein is a capsid protein of an icosahedral virus that forms a pentamer in a small unit during the self-assembly process.

11. The method for producing a target protein fusion pentamer according to claim 8, wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus.

12. The method for producing a target protein fusion pentamer according to claim 8, wherein the viral nucleocapsid contains the amino acid sequence of SEQ ID NO:

1.

13. The method for producing a target protein fusion pentamer according to claim 8, wherein the host cell is Escherichia coli.

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