Platform for generating target protein fused pentamers based on pentamer toxin proteins using viral nucleocapsids

Abstract

Expression vectors of the present invention for producing a target protein fused pentamer comprise a polynucleotide encoding a pentamer toxin protein as a fusion partner for a target protein and a viral nucleocapsid. In addition, the method for producing a target protein-fused pentamer of the present invention comprises: producing an expression vector for producing a target protein-fused pentamer, the expression vector comprising a polynucleotide encoding a target protein and a pentamer toxin protein and a viral nucleocapsid which are fusion partners for the target protein; producing a transformant by introducing the expression vector for producing a pentamer fused with a target protein into a host cell; and culturing the transformant.

Description

Technical Field

[0001] The present invention relates to a platform for producing pentamers of target protein fusion based on pentamer toxin protein using viral nucleocapsid. More particularly, the present invention relates to an expression vector, a host cell transformed by the expression vector, and a method for producing pentamers of target protein fusion using the expression vector, wherein the expression vector uses pentamer toxin protein and viral nucleocapsid as fusion partners, and can effectively produce pentamer target protein in host cells. Background Art

[0002] Although there are several systems for producing proteins, it is preferred to use prokaryotic cell systems such as Escherichia coli for rapid, low-cost large-scale production if possible. However, in these systems, there is a problem that many proteins, particularly proteins derived from eukaryotic cells or viral proteins that cause infection, lose their solubility and become insoluble precipitates.

[0003] Meanwhile, in order to improve vaccine efficacy, technologies for producing antigen proteins in a multimeric form are being studied, and one of these technologies is a method using a toxin protein that forms a pentameric structure. Toxin proteins belonging to the heat-labile enterotoxin (LT) family are known to form such a pentameric structure, and among these toxin proteins, cholera toxin B subunit (CTB) can be used as a carrier function to promote the absorption of chemically or genetically related exogenous antigens through the mucosa.

[0004] Although attempts have been made to produce such pentamer toxin protein-based fusion proteins in prokaryotic cell systems such as E. coli for rapid and low-cost large-scale production, they are produced as insoluble precipitates due to problems associated with protein folding, and even if they undergo refolding, it is difficult to produce the desired pentamer. Therefore, it is necessary to develop technologies for solving these problems. Summary of the invention

[0005] Technical issues

[0006] The present invention relates to an expression vector which can effectively produce a target protein in pentamer form, in particular a difficult-to-express protein (DtEP) which is insoluble in host cells, especially Escherichia coli.

[0007] The present invention also provides a host cell transformed by the expression vector to effectively produce the pentamer.

[0008] The present invention also provides a method for efficiently producing pentamers using an expression vector and / or a host cell.

[0009] Technical Solution

[0010] The present invention provides an expression vector for producing a pentamer fused with a target protein, the expression vector comprising a polynucleotide encoding a pentamer toxin protein and a viral nucleocapsid as a fusion partner of the target protein.

[0011] The present invention also provides a host cell transformed by an expression vector for producing a pentamer fused with a target protein.

[0012] The present invention also provides a method for producing a target protein fused pentamer, the method comprising manufacturing an expression vector for producing the target protein fused pentamer, the expression vector comprising a polynucleotide encoding the target protein and a pentamer toxin protein and a viral nucleocapsid as fusion partners of the target protein; producing a transformant by introducing the expression vector for producing the target protein fused pentamer into a host cell; and culturing the transformant.

[0013] Beneficial Effects

[0014] According to the present invention, target proteins, particularly difficult-to-express proteins (DtEP) that are insoluble in E. coli, can be efficiently produced in the form of pentamers in host cells, particularly E. coli. In particular, according to the present invention, vaccine antigens with greatly improved immunogenicity can be efficiently produced by forming fine pentamers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Fusion protein constructs of expression vectors used in the experiments are shown, including an expression vector according to one embodiment of the present invention.

[0016] Figure 2 The expression of Figure 1 SDS-PAGE results of fusion protein construct 1.

[0017] Figure 3 The expression of Figure 1 SDS-PAGE results of fusion protein construct 2.

[0018] Figure 4 The expression of Figure 1 SDS-PAGE results of fusion protein construct 3.

[0019] Figure 5 The expression of Figure 1 SDS-PAGE results of fusion protein construct 5.

[0020] Figure 6 The expression of Figure 1 SDS-PAGE results of fusion protein construct 6.

[0021] Figure 7 and Figure 8 Shows Figure 1 Results of GM1 binding assay of the expression products (pentamers) of each fusion protein construct. DETAILED DESCRIPTION

[0022] According to one aspect of the present invention, an expression vector for producing a pentamer fused with a target protein is provided, the expression vector comprising a polynucleotide encoding a pentamer toxin protein and a viral nucleocapsid as a fusion partner of the target protein.

[0023] The term "target protein" used herein refers to a protein to be produced in large quantities by a person of ordinary skill in the art, including all proteins that can be expressed in a host cell after a polynucleotide encoding the protein is inserted into a recombinant vector.

[0024] In the present invention, the target protein may be selected from antigens, antibodies, cell receptors, enzymes, structural proteins, serum and cell proteins, but the present invention is not limited thereto.

[0025] In the present invention, the target protein may be a viral antigen protein that induces an immune response. For example, the target protein may be an antigen protein of a virus having a spherical icosahedral capsid structure, and the icosahedron is a structure formed by 20 equilateral triangles, i.e., subunits, and composed of 12 vertices, and there are mainly two-fold axes, three-fold axes, and five-fold axes. The target protein may be an antigen protein of a virus in which subunits are combined in the form of pentagons or hexagons according to the number of axes.

[0026] In the present invention, the target protein can be a capsid protein of an icosahedral virus that forms a pentamer (or hexamer) with a subunit during self-assembly. When the protein is used as a target protein, a recombinant pentamer having the same level of antigenicity as the pentamer of the original viral capsid protein can be produced, particularly in Escherichia coli.

[0027] As target protein, generally, any pentameric antigen of enveloped or non-enveloped viruses formed in an icosahedron can be used.

[0028] Examples of enveloped viruses having a lipid membrane may include viruses of the Herpesviridae and Hepadnaviridae families having a DNA genotype, and viruses of the Flaviviridae family having an RNA genotype.

[0029] Examples of non-enveloped viruses or naked viruses composed of viral envelope proteins without a lipid membrane may include viruses of the Adenoviridae and Parvoviridae families having a DNA genotype, and viruses of the Reoviridae, Picornaviridae, and Caliciviridae families having an RNA genotype.

[0030] The term "expression vector" as used herein is a vector for expressing a polynucleotide encoding a polypeptide of interest by introducing it into a heterologous or homologous host cell, and refers to a circular or linear polynucleotide, e.g., a DNA or RNA molecule. An expression vector may include a promoter and / or terminator sequence, and may also include components required for use in the vector, such as an origin of replication, a selectable marker, a polyadenylation signal, etc.

[0031] The expression vector of the present invention may be a plasmid, a viral vector, a phage particle or a genomic insert, and may be replicated regardless of the genome of the host cell or integrated into the genome of the host cell after being introduced into the host cell.

[0032] The expression vector of the present invention may include a polynucleotide encoding a target protein, or may be used to include a polynucleotide encoding a target protein in the future. In particular, in the latter case, as a component allowing easy inclusion of a target protein encoding polynucleotide, a restriction enzyme recognition site generally referred to as a multiple cloning site (MSC) is preferably included.

[0033] The present invention is based on new research results: when pentameric toxin protein and viral nucleocapsid are used as fusion partners of target protein, the target protein can be effectively expressed (for example, a protein that is insoluble when no fusion partner is used, or a protein that can be soluble when another existing fusion partner is used); and when pentameric toxin protein and viral nucleocapsid are used as fusion partners of target protein, pentamers in which the target protein self-assembles can be effectively produced.

[0034] When such fusion partners are used, there is the advantage of producing a target protein with increased solubility, and generally speaking, the larger the size of the fusion partner, the more helpful it is in increasing the solubility of the target protein in water. However, increased structural interference with adjacent target proteins due to the size of the fusion partner may also hinder the generation of physiologically active structures. Therefore, it is very important to find a fusion partner that can increase the solubility and activity of the target protein despite its small size, if possible.

[0035] In particular, in the present invention, the viral nucleocapsid used as a fusion partner has a very small size (short peptide), and therefore has the advantages of minimizing structural interference with the target protein and minimizing structural interference with the self-assembly of the target protein monomers (self-assembly into pentamers in the present invention).

[0036] In addition, according to the present invention, there is an advantage of utilizing the zinc binding domain of the viral nucleocapsid. For example, by utilizing the zinc binding domain of the viral nucleocapsid, such as a zinc affinity purification method, the fusion protein can be easily purified without the need for a separate component for purification, such as a histidine tag. In the present invention, the viral nucleocapsid can be a conventionally known viral nucleocapsid, and it can be a fragment or variant of the viral nucleocapsid, as long as the activity of the present invention is retained, in other words, as long as the activity of forming pentamers is retained, wherein the viral nucleocapsid is used as a fusion partner of the target protein together with the pentamer toxin protein to allow the target protein to be soluble, particularly in Escherichia coli.

[0037] In the present invention, the viral nucleocapsid may be a nucleocapsid of a retrovirus, such as Human Immunodeficiency Virus-1 (HIV-1), SIV or MuLV.

[0038] In the present invention, the viral nucleocapsid is preferably the nucleocapsid of human immunodeficiency virus (HIV).

[0039] In the present invention, the viral nucleocapsid may include an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 1. The viral nucleocapsid preferably includes an amino acid sequence that retains amino acids 14, 17, 22, and 27, more preferably amino acids 14, 17, 22, 27, 35, 38, 43, and 48, and even more preferably amino acids 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48, and 51 of SEQ ID NO: 1 and satisfies the above sequence identity.

[0040] In the present invention, the polynucleotide encoding the viral nucleocapsid includes a nucleotide sequence having preferably 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2 or 3, or the nucleotide sequence of SEQ ID NO: 2 or 3. The polynucleotide encoding the viral nucleocapsid includes a nucleotide sequence encoding the following amino acid sequence and satisfies the above sequence identity: the amino acid sequence preferably retains amino acids 14, 17, 22 and 27 of SEQ ID NO: 1, more preferably amino acids 14, 17, 22, 27, 35, 38, 43 and 48, and even more preferably amino acids 6, 9, 10, 13, 14, 17, 19, 22, 25, 27, 28, 31, 32, 33, 35, 37, 38, 40, 43, 46, 48 and 51.

[0041] Sequence identity used herein 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 position of the sequence being compared. When the position of the comparison sequence is occupied by the same amino acid or base, the molecules are identical at that position. The degree of identity between an amino acid or nucleotide sequence is a function of the quantity of the same amino acid or nucleotide at each position that is common to the same amino acid or nucleotide sequence. For example, using a variety of alignment algorithms and / or programs (including FASTA or BLAST), the identity between two sequences can be calculated.

[0042] In the present invention, pentameric toxin protein is a toxin protein as a whole and has the activity of forming a composite protein with a pentameric shape of five subunits. As long as the activity of forming a pentameric composite protein is maintained, conventionally known pentameric toxin proteins and fragments of pentameric toxin proteins can be used. For example, pentameric toxin protein can be selected from cholera toxin B subunit (CTB), Escherichia coli heat-labile enterotoxin B subunit (LTB) and Shiga toxin B subunit.

[0043] In the present invention, the pentameric toxin protein preferably includes an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the amino acid sequence of SEQ ID NO:4 or 6, or the amino acid sequence of SEQ ID NO:4 or 6.

[0044] In the present invention, the polynucleotide encoding the pentameric toxin protein preferably includes a nucleotide sequence having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence identity with the nucleotide sequence of SEQ ID NO: 5 or 7, or the nucleotide sequence of SEQ ID NO: 5 or 7.

[0045] The expression vector of the present invention can be used to produce the target protein fused pentamer in cells selected from all organisms including prokaryotes and eukaryotes, preferably in microorganisms of the genus Escherichia, and more preferably in Escherichia coli.

[0046] In the expression vector of the present invention, the polynucleotide encoding the pentameric toxin protein and the viral nucleocapsid may also include other components, for example, a cloning site for inserting the target protein encoding polynucleotide, such as a restriction enzyme recognition site, such as a multiple cloning site (MCS); a linker sequence for providing a gap 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, such as a histidine tag sequence; and / or a protease recognition site coding sequence for cutting a portion of the expressed fusion protein. If desired, each component may be arranged in various ways, or the number of these components may be changed.

[0047] 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, the polynucleotide encoding the pentameric toxin protein and the viral nucleocapsid connected to the N-terminus of the pentameric toxin protein, and includes a target protein coding site or a cloning site for connecting the target protein to the C-terminus of the pentameric toxin protein. As a specific example of 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 encoding polynucleotide or a cloning site for inserting a target protein coding site (e.g., a restriction enzyme recognition site, e.g., MCS) and a terminator are operably connected in sequence along the 5' to 3' direction.

[0048] 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, the polynucleotide encoding the pentameric toxin protein and the viral nucleocapsid connected to the C-terminus of the pentameric toxin protein, and including a target protein coding site or a cloning site for connecting the target protein coding site between the pentameric toxin protein and the viral nucleocapsid. As a specific example of an embodiment, the polynucleotide includes a construct in which a promoter, a viral nucleocapsid coding site, a target protein coding site or a cloning site for inserting a target protein coding polynucleotide (e.g., a restriction enzyme recognition site, e.g., MCS), a viral nucleocapsid coding site, and a terminator are operably connected in sequence along the 5' to 3' direction.

[0049] The expression vector of the present invention may include a T7 promoter and a T7 terminator to regulate the transcription of the DNA encoding the target protein and the fusion partner through the bacteriophage T7 system. Accordingly, the expression vector of the present invention can more efficiently produce the target protein in Escherichia coli.

[0050] The expression vector of the present invention can be produced using conventional recombinant methods based on, for example, conventional expression vectors.

[0051] According to another aspect of the present invention, a host cell transformed by the expression vector of the present invention for producing a target protein fused pentamer is provided.

[0052] The host cell in the present invention can be selected from cells of all organisms including prokaryotes and eukaryotes, preferably selected from cells of microorganisms of the genus Escherichia, more preferably selected from cells of Escherichia coli.

[0053] The term "conversion" or "introduction" as used herein means that a polynucleotide is introduced into a host so that the polynucleotide can be replicated as an extrachromosomal factor or by completing chromosomal integration. The transformation method using the expression vector according to the present invention may include 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, but the present invention is not limited thereto.

[0054] According to another aspect of the present invention, a method for producing a target protein fused pentamer includes manufacturing an expression vector for producing a target protein fused pentamer, the expression vector including a polynucleotide encoding the target protein and a pentamer toxin protein and a viral nucleocapsid as fusion partners of the target protein; producing a transformant by introducing the expression vector for producing the target protein fused pentamer into a host cell; and culturing the transformant.

[0055] Here, for specific details such as matters concerning the expression vector and matters concerning introduction of the expression vector, reference can be made to the matters described previously.

[0056] In order to cultivate the transformant, any culture method known to be suitable for the host cell can be used. For example, when the host is Escherichia coli, a method of culturing in Luria-Bertani (LB) medium at 16°C to 37°C can be used. If there is a component of an antibiotic selection marker in the expression vector (for example, a gene resistant to the antibiotic ampicillin), then at this time, by adding the corresponding antibiotic (for example, ampicillin) to the culture medium and culturing the cells, the proliferation of the Escherichia coli containing the expression vector can be preferentially induced. In addition, when the expression vector contains a component (for example, lac operon-related component) that regulates the transcription and / or translation of the DNA encoding the target protein, in the expression vector, the expression of the target protein can be induced by adding related materials (such as IPTG).

[0057] The following examples are presented to help understand the present invention and the following contents do not limit the scope of the present invention.

[0058] Example

[0059] Preparation Example 1

[0060] To investigate the suitability of a combination of a pentameric toxin protein and a viral nucleocapsid (NC) as a fusion partner for the production of pentamers, a vector was constructed in which the NC of human immunodeficiency virus (HIV) was linked to the N-terminus of the Escherichia coli heat-labile enterotoxin B subunit (LTB) as a pentameric toxin protein and a six-histidine tag was linked to the C-terminus of LTB ( Figure 1 Construct 1).

[0061] Here, the vector was manufactured based on the pGEMEX-1 (Promega) vector designed for regulating target protein expression by 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.

[0062] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:8, and the amino acid sequence of the expected expressed fusion protein is identical to SEQ ID NO:9.

[0063] Example 1

[0064] A pentamer vector for producing a fusion protein was prepared in the form in which NC was linked to the N-terminus of LTB as a pentamer toxin protein, a tobacco etch virus (TEV) protease cleavage site and a six-histidine tag were present therebetween, and VP1 of poliovirus as a target protein was linked to the C-terminus of LTB ( Figure 1 Construct 2).

[0065] As described in Preparation Example 1, this vector was produced based on the pGEMEX-1 (Promega) vector.

[0066] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:10, and the amino acid sequence of the expected expressed fusion protein is identical to SEQ ID NO:11.

[0067] Example 2

[0068] A pentamer vector for producing a fusion protein was manufactured in the form in which NC was linked to the N-terminus of cholera toxin B subunit (CTB) of Bacillus cholerae as a pentameric toxin protein, a TEV protease cleavage site and a six-histidine tag were present therebetween, and VP1 of poliovirus as a target protein was linked to the C-terminus of CTB ( Figure 1 Construct 3).

[0069] As described in Preparation Example 1, this vector was produced based on the pGEMEX-1 (Promega) vector.

[0070] The sequence of the construct region in the manufactured vector was identical to SEQ ID NO:12, and the amino acid sequence of the expected expressed fusion protein was identical to SEQ ID NO:13.

[0071] Comparative Example 1

[0072] A pentamer vector for producing a fusion protein was manufactured in the form in which EPRS was linked to the N-terminus of LTB as a pentamer toxin protein instead of NC, a tobacco etch virus (TEV) protease cleavage site and a six-histidine tag were present therebetween, and VP1 of poliovirus as a target protein was linked to the C-terminus of LTB ( Figure 1 Construct 4).

[0073] Here, EPRS includes the WHEP domain of human-derived glutamyl-prolyl-tRNA synthetase (a WHEP domain located in the middle of EPRS, which includes a linker connecting TRS-1, TRS-2, and TRS-3 to the three domains).

[0074] As described in Preparation Example 1, this vector was produced based on the pGEMEX-1 (Promega) vector.

[0075] Example 3

[0076] A pentamer vector for producing a fusion protein was prepared in the form of NC linked to the N-terminus of LTB as a pentamer toxin protein, six histidine tags and a TEV protease cleavage site were present therebetween, and VP1 of foot-and-mouth disease virus (FMDV) as a target protein was linked to the C-terminus of LTB ( Figure 1 Construct 5).

[0077] As described in Preparation Example 1, this vector was produced based on the pGEMEX-1 (Promega) vector.

[0078] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:14, and the amino acid sequence of the expected expressed fusion protein is identical to SEQ ID NO:15.

[0079] Example 4

[0080] A pentamer vector for producing a fusion protein was prepared in the form in which VP1 of FMDV as a target protein was linked to the C-terminus of LTB as a pentamer toxin protein, NC was linked to the C-terminus of FMDV VP1, and a six-histidine tag was linked to the C-terminus of NC ( Figure 1 Construct 6).

[0081] As described in Preparation Example 1, this vector was produced based on the pGEMEX-1 (Promega) vector.

[0082] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:16, and the amino acid sequence of the expected expressed fusion protein is identical to SEQ ID NO:17.

[0083] Comparative Example 2

[0084] A pentamer vector for producing a fusion protein was manufactured in the form in which EPRS was linked to the N-terminus of LTB as a pentamer toxin protein instead of NC, a six-histidine tag and a TEV protease cleavage site were present therebetween, and VP1 of FMDV as a target protein was linked to the C-terminus of LTB ( Figure 1 Construct 7).

[0085] As described in Preparation Example 1, this vector was produced based on the pGEMEX-1 (Promega) vector.

[0086] Experimental Examples

[0087] The vectors of the Preparation Example, Example and Comparative Example were transformed into E. coli Shuffle / T7 / pLysS and cultured. Shuffle / T7 / pLysS was prepared in the following manner: pLysS OneShot was transformed from BL21 Star (DE3) by Mini-prep TM pLysS plasmid was extracted from competent E. coli (Invitrogen), the extracted pLysS was introduced into Shuffle T7 (NEB) competent E. coli, and selected with chloramphenicol (CM), an antibiotic marker included in pLysS. All transformed E. coli were incubated in LB medium containing 50 μg / mL ampicillin and 34 μg / mL CM. The culture temperature was set to 16°C to 37°C. When the OD600 value of E. coli reached 0.5 or more, in order to activate the T7 promoter, 0 μM to 1 mM IPTG was added, and in order to fully produce protein, the E. coli cells were cultured at 30°C for about 3 hours or at 16°C to 20°C for about 16 hours after adding IPTG. The fully cultured E. coli was centrifuged to remove the supernatant and then stored. Subsequently, 0.3 mL of PBS was added to the E. coli harvest corresponding to 5 mL of LB medium, and it was subjected to ultrasonic treatment to obtain a lysate. Then, the lysate was centrifuged and separated into a soluble fraction and a precipitate fraction, and the total lysate, soluble fraction and precipitate fraction were analyzed by SDS-PAGE, respectively.

[0088] First, if Figure 2 As shown, as a result of inducing the expression of the fusion protein by introducing the vector of Preparation Example 1 into E. coli, a high level of fusion protein was normally expressed, i.e., with the expected size and solubility. This indicates that the combination of pentameric toxin protein and NC has the potential to be used as a fusion partner for producing pentamers fused to the target protein.

[0089] like Figure 3 As shown, as a result of inducing the expression of the fusion protein by introducing the vector of Example 1 into E. coli, a high level of the fusion protein was normally expressed, i.e., with the expected size and solubility. In particular, it is known that LTB is insoluble in E. coli, making it very difficult to be expressed normally. Therefore, these results show that the method of the present invention can express a protein previously insoluble in E. coli as soluble.

[0090] like Figure 4As shown, as a result of inducing the expression of the fusion protein by introducing the vector of Example 2 into Escherichia coli, the fusion protein was normally expressed at a high level.

[0091] like Figure 5 As shown, as a result of inducing the expression of the fusion protein by introducing the vector of Example 3 into Escherichia coli, the fusion protein was normally expressed at a high level.

[0092] like Figure 6 As shown, as a result of inducing the expression of the fusion protein by introducing the vector of Example 4 into Escherichia coli, a high level of the fusion protein was normally expressed. In this case, since the arrangement of the pentameric toxin protein, the target protein, and the NC was changed, these results indicate that normal expression is possible even when the arrangement of each component is changed.

[0093] In addition, the GM1 binding affinity of the product (i.e., pentamer) is studied by inducing the expression of each of the vectors of the preparation examples, the examples and the comparative examples. The toxin protein belonging to the enterotoxin family shows the ability to form pentamers on the surface of the intestinal mucosa and to bind to the GM1 ganglioside receptor. Therefore, the fact that the expression product can bind to the GM1 ganglioside means that the pentamer is well formed, and the protein is appropriately constructed, rather than simply expressed in a soluble form. Accordingly, this point has been confirmed by studying the GM1 binding affinity.

[0094] Specifically, GM1 was diluted in bicarbonate buffer (pH 9.6), distributed in an immunoplate (Maxisorp, Thermo Scientific), and coated at 4°C for about 16 hours. Subsequently, the plate was washed three times with PBS-T (Tween-20 0.1%), and the expression product sample was diluted 2 times (the initial dilution concentration was set to 100nM, and the sample was serially diluted), added to each well, and allowed to react at room temperature for 4 hours to allow sufficient binding to GM1. Then, the anti-6His tag as the first antibody was diluted 1:2,000 in serum dilution buffer (PBS-T containing 0.25% BSA), added to each well of the plate, and allowed to react at room temperature for 1 hour. Then, the anti-mouse (HRP-conjugated) as the second antibody was diluted 1:10,000 in serum dilution buffer, added to each well of the plate, and allowed to react at room temperature for 1 hour. After reacting with the primary antibody and the secondary antibody, the cells were washed with PBS-T, and after reacting with the secondary antibody, TMB (BD OptEIA TMB substrate reagent set) was added to each well to induce color development of HRP. After 30 minutes, the reaction was terminated by adding 2N sulfuric acid, and the resulting product was analyzed using an ELISA plate reader.

[0095] like Figure 7As shown, as a result of studying the GM1 binding affinity of Preparation Example 1, Example 1, Example 2 and Comparative Example 1, it has been shown that Example exhibits superior GM1 binding affinity than Preparation Example 1 and Comparative Example 1.

[0096] like Figure 8 As shown, as a result of studying the GM1 binding affinity of Preparation Example 1, Example 3, Example 4 and Comparative Example 2, it has been shown that Examples exhibited superior GM1 binding affinity than Preparation Example 1 and Comparative Example 2.

[0097] In the above, the present invention is described with reference to the embodiments. It will be appreciated by those skilled in the art that the present invention can be implemented in a modified form without departing from the basic features of the present invention. Therefore, the disclosed embodiments should be considered from a descriptive perspective rather than a restrictive perspective. The scope of the present invention is shown in the claims rather than in the above description, and all differences within their equivalent ranges will be interpreted as being included in the present invention.

[0098] < Sequence Listing >

[0099] SEQ ID NO:1

[0100] Sequence name: HIV nucleocapsid amino acid sequence

[0101] Sequence Type: AA

[0102] Name of Organism: Human Immunodeficiency Virus

[0103] sequence:

[0104] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQ MKDCTERQAN

[0105] SEQ ID NO:2

[0106] Sequence name: Nucleotide sequence encoding HIV nucleocapsid

[0107] Sequence type: DNA

[0108] Name of Organism: Human Immunodeficiency Virus

[0109] sequence:

[0110] CAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAAC

[0111] SEQ ID NO:3

[0112] Sequence name: Nucleotide sequence encoding HIV nucleocapsid

[0113] Sequence type: DNA

[0114] Organism Name: Synthetic Construct

[0115] sequence:

[0116] CAGCGTGGTAACTTCCGTAACCAGCGTAAAACCGTTAAATGCTTCAACTGCGGCAAAGAAGGCCACATCGCGAAAAACTGCCGTGCGCCGCGTAAAAAAGGCTGCTGGCGTTGCGGCCGTGAAGGCCACCAGATGAAAGATTGCACCGAACGTCAGGCGAAC

[0117] SEQ ID NO:4

[0118] Sequence name: Amino acid sequence of heat-labile enterotoxin B subunit from Escherichia coli Sequence type: AA

[0119] Organism Name: Escherichia coli

[0120] sequence:

[0121] APQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKN

[0122] SEQ ID NO:5

[0123] Sequence name: Nucleotide sequence encoding heat-labile enterotoxin B subunit from Escherichia coli Sequence type: DNA

[0124] Organism Name: Escherichia coli

[0125] sequence:

[0126] GCTCCCCAGACTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGAAACATTTCAGGTCGAAG TCCCGGGCAGTCAACATATAGACTCCCAGAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGAAAAAC

[0127] SEQ ID NO:6

[0128] Sequence name: Amino acid sequence of cholera toxin B subunit from Vibrio cholerae Sequence type: AA

[0129] Name of Organism: Vibrio cholerae

[0130] sequence:

[0131] TPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNGATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN

[0132] SEQ ID NO:7

[0133] Sequence name: Nucleotide sequence encoding cholera toxin B subunit from Vibrio cholerae Sequence type: DNA

[0134] Name of Organism: Vibrio cholerae

[0135] sequence:

[0136] ACTCCGCAGAACATTACGGACCTGTGTGCGGAGTATCATAATACGCAGATTCACACTTTGAATGACAAGATTTTTTCATATACGGAGTCATTAGCTGGTAAACGTGAAATGGCAATTATCACTTTTAAAAATGGTGCGACGTTCCAGGTGGAAGTTCCGGGCAGTCAGCATATTGATAGTCAGAAAAAAGCCATCGAACGTATGAAGGATACCTTGCGTATTGCGTACTTAACCGAGGCTAAAGTCGAGAAATTATGTGTCTGGAATAATAAGACCCCACATGCCATTGCTGCGATTTCGATGGCCAAT

[0137] SEQ ID NO:8

[0138] Sequence Name: Nucleotide sequence of the fusion protein construct

[0139] Sequence Type: DNA

[0140] Organism Name: Synthetic construct

[0141] Sequence:

[0142] ATGCAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAACGGATCCGCTCCCCAGACTATTACAGAACTATGTTCGGAATATCGCAACACACAAATATATACGATAAATGACAAGATACTATCATATACGGAATCGATGGCAGGCAAAAGAGAAATGGTTATCATTACATTTAAGAGCGGCGAAACATTTCAGGTCGAAGTCCCGGGCAGTCAACATATAGACTCCCAGAAAAAAGCCATTGAAAGGATGAAGGACACATTAAGAATCACATATCTGACCGAGACCAAAATTGATAAATTATGTGTATGGAATAATAAAACCCCCAATTCAATTGCGGCAATCAGTATGAAAAAC

[0143] SEQ ID NO:9

[0144] Sequence name: amino acid sequence of the expected fusion protein

[0145] Sequence Type: AA

[0146] Organism Name: Synthetic Construct

[0147] sequence:

[0148] MQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANGSAPQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKN

[0149] SEQ ID NO:10

[0150] Sequence name: Nucleotide sequence of fusion protein construct

[0151] Sequence type: DNA

[0152] Organism Name: Synthetic Construct

[0153] sequence:

[0154]

[0155] SEQ ID NO:11

[0156] Sequence name: amino acid sequence of the expected fusion protein

[0157] Sequence Type: AA

[0158] Organism Name: Synthetic Construct

[0159] sequence:

[0160] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDDGTGSAPQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSG ETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNDIGLGQMLESMIDNTVRETVGAATSRDALPNTEASGPTHSKEIPALTAVETGATNPLVPSDTV QTRHVVQHRSRSESSIESFFARGACVTIMTVDNPASTTNKDKLFAVWKITYKDTVQLRRKLEFFTYSRFDMELTFVVTANFTETNNGHALNQVYQIMYVPPGAPVPEKWDDYTWQTSSNP SIFYTYGTAPARISVPYVGISNAYSHFYDGFSKVPLKDQSAALGDSLYGAASLNDFGILAVRVVNDHNPTKVTSKIRVYLKPKHIRVWCRPRPRAVAYYGPGVDYKDGTLTPLSTKDLTTY

[0161] SEQ ID NO:12

[0162] Sequence name: Nucleotide sequence of fusion protein construct

[0163] Sequence type: DNA

[0164] Organism Name: Synthetic Construct

[0165] sequence:

[0166]

[0167] SEQ ID NO:13

[0168] Sequence name: amino acid sequence of the expected fusion protein

[0169] Sequence Type: AA

[0170] Organism Name: Synthetic Construct

[0171] sequence:

[0172] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDDGTGSTPQNITDLCAEYHNTQIHTLNDKIFSYTESLAGKREMAIITFKNG ATFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANDIGLGQMLESMIDNTVRETVGAATSRDALPNTEASGPTHSKEIPALTAVETGATNPLVPSDTV QTRHVVQHRSRSESSIESFFARGACVTIMTVDNPASTTNKDKLFAVWKITYKDTVQLRRKLEFFTYSRFDMELTFVVTANFTETNNGHALNQVYQIMYVPPGAPVPEKWDDYTWQTSSNP SIFYTYGTAPARISVPYVGISNAYSHFYDGFSKVPLKDQSAALGDSLYGAASLNDFGILAVRVVNDHNPTKVTSKIRVYLKPKHIRVWCRPRPRAVAYYGPGVDYKDGTLTPLSTKDLTTY

[0173] SEQ ID NO:14

[0174] Sequence name: Nucleotide sequence of fusion protein construct

[0175] Sequence type: DNA

[0176] Organism Name: Synthetic Construct

[0177] sequence:

[0178]

[0179] SEQ ID NO:15

[0180] Sequence name: amino acid sequence of the expected fusion protein

[0181] Sequence Type: AA

[0182] Organism Name: Synthetic Construct

[0183] sequence:

[0184] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDGTGSAPQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNDITTTTGESADPVTTTV ENYGGETQTARRLHTDVAFVLDRFVKLTQPKSTQTLDLMQIPSHTLVGALLRSATYYFSDLEVALVHTGPVTWVPNGAPKTALNNHTNPTAYQKQPITRLALPYTAPHRVLSTVYNGKTTYGEESSRRGDLAALARRVNNRLPTSFNYGAVKADTITELLIRMKRAETYCPRPLLALDTTQDRRKQKIIAPEKQMI

[0185] SEQ ID NO:16

[0186] Sequence name: Nucleotide sequence of fusion protein construct

[0187] Sequence type: DNA

[0188] Organism Name: Synthetic Construct

[0189] sequence:

[0190]

[0191] SEQ ID NO:17

[0192] Sequence name: amino acid sequence of the expected fusion protein

[0193] Sequence Type: AA

[0194] Organism Name: Synthetic Construct

[0195] sequence:

[0196] APQTITELCSEYRNTQIYTINDKILSYTESMAGKREMVIITFKSGETFQVEVPGSQHIDSQKKAIERMKDTLRITYLTETKIDKLCVWNNKTPNSIAAISMKNGSTTTTGESADPVTTTVENYGGETQTARRLHTDVAFVLDRFVKLTQPKSTQTLDLMQIPSHTLVGALLRSATYYFSDLEVALVHTGP VTWVPNGAPKTALNNHTNPTAYQKQPITRLALPYTAPHRVLSTVYNGKTTYGEESSRRGDLAALARRVNNRLPTSFNYGAVKADTITELLIRMKRAETYCPRPLLALDTTQDRRKQKIIAPEKQMIDIQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANKLHHHHHH

Claims

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

2. The expression vector according to claim 1, wherein the pentameric toxin protein is selected from cholera toxin B subunit (CTB), Escherichia coli heat-labile enterotoxin B subunit (LTB) and Shiga toxin B subunit.

3. The expression vector according to claim 1, wherein the target protein is a capsid protein of an icosahedral virus that forms a pentamer with subunits during self-assembly. The expression vector according to claim 1 , wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus.

5. The expression vector according to claim 1, wherein the viral nucleocapsid comprises the amino acid sequence of SEQ ID NO:

1. The expression vector according to claim 1 , wherein the vector is used to produce a pentamer fused with a target protein in Escherichia coli .

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

8. A method for producing a target protein fused pentamer, the method comprising: Manufacturing an expression vector for producing a pentamer fused with a target protein, the expression vector comprising a polynucleotide encoding the target protein and a pentamer toxin protein and a viral nucleocapsid as fusion partners of the target protein; Producing a transformant by introducing the expression vector for producing the target protein fused pentamer into a host cell; as well as The transformants were cultured.

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

10. The method of claim 8, wherein the target protein is a capsid protein of an icosahedral virus that forms a pentamer with subunits during self-assembly.

11. The method of claim 8, wherein the viral nucleocapsid is a nucleocapsid of a human immunodeficiency virus.

12. The method of claim 8, wherein the viral nucleocapsid comprises the amino acid sequence of SEQ ID NO:

1.

13. The method of claim 8, wherein the host cell is Escherichia coli.

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