Platform for producing self-assembled nanoparticles fused with target proteins based on crystallizable proteins using viral nucleocapsids

Abstract

Expression vectors for the production of target protein fused self-assembled nanoparticles of the present invention include polynucleotides encoding a crystallizable protein as a fusion partner for a target protein and a viral nucleocapsid. Further, the method of producing target protein fused self-assembled nanoparticles of the present invention comprises making an expression vector for producing target protein fused self-assembled nanoparticles, the expression vector comprising a polynucleotide encoding a target protein and a crystallizable protein and a viral nucleocapsid as fusion partners for the target protein; producing a transformant by introducing the expression vector for producing the self-assembled nanoparticles fused with the target protein into a host cell; and culturing the transformant.

Description

Technical Field

[0001] The present invention relates to a platform for producing self-assembled nanoparticles fused with a target protein based on a crystallizable protein using a viral nucleocapsid. More specifically, the present invention relates to an expression vector that can effectively produce self-assembled nanoparticles, which are fused with a target protein, particularly a difficult-to-express protein (DtEP) that is insoluble and expressed in Escherichia coli, and present the target protein on the surface in a host cell, particularly Escherichia coli, for example, in an immunologically significant structure using a crystallizable protein and a viral nucleocapsid as a fusion partner, the present invention relates to a host cell transformed with the expression vector, and a method for producing self-assembled nanoparticles fused with a target protein using the expression vector. Background Art

[0002] In the vaccine technology used to deal with infectious diseases, a chimeric VLP (virus-likeparticle, virus-like particle) technology has been recently developed, which is used to introduce exogenous pathogenic antigens into a virus-like particle (VLP) structure that is well formed into a polymer structure, and has been used to develop vaccines targeting various pathogens. This chimeric VLP presents exogenous pathogenic antigens on the surface of a VLP-based structure composed of structural proteins or self-assembling proteins of another specific virus.

[0003] Use chemical conjugation or gene fusion method to introduce exogenous antigen into the structure based on VLP.Chemical conjugation method chemically fuses the exogenous antigen prepared separately to the structure based on VLP prepared by covalent bonding, but has the disadvantages of difficulty in arranging antigens in a specific direction and low density of presenting antigens.On the other hand, gene fusion method has the following advantages: by fusion expression of VLP structure forming protein gene and target antigen protein gene at DNA level, chimeric VLP can be prepared by simpler method.However, this method has the problem of limited exogenous antigen that can be introduced, especially when Escherichia coli expression system is used for efficient and rapid production, due to lack of post-translational molecular chaperone or processing, such as the abnormal protein expression problem caused by forming insoluble protein aggregates.Therefore, it is necessary to develop technology to solve the above problems. Summary of the invention

[0004] Technical issues

[0005] The present invention aims to provide an expression vector that can effectively produce self-assembled nanoparticles that are fused with a target protein, especially a difficult-to-express protein (DtEP) that is insoluble in Escherichia coli, and present the target protein on the surface of a host cell, especially Escherichia coli, for example, in an immunologically significant structure.

[0006] The present invention also provides a host cell, which is transformed with an expression vector to effectively produce self-assembled nanoparticles.

[0007] The present invention also provides a method for efficiently producing self-assembled nanoparticles using the expression vector and / or the host cell.

[0008] Technical Solution

[0009] The present invention provides an expression vector for producing self-assembling nanoparticles fused with a target protein, wherein the expression vector comprises a polynucleotide encoding a crystallizable protein and a viral nucleocapsid as a fusion partner of the target protein.

[0010] The present invention also provides a host cell, which is transformed with an expression vector to produce self-assembled nanoparticles fused with a target protein.

[0011] The present invention also provides a method for producing self-assembled nanoparticles fused with a target protein, the method comprising manufacturing an expression vector for producing self-assembled nanoparticles fused with a target protein, the expression vector comprising a polynucleotide encoding a target protein and a crystallizable protein and a viral nucleocapsid as fusion partners of the target protein; producing a transformant by introducing the expression vector for producing self-assembled nanoparticles fused with a target protein into a host cell; and culturing the transformant.

[0012] Beneficial Effects

[0013] According to the present invention, self-assembled nanoparticles can be efficiently produced in host cells, especially Escherichia coli, which are fused with target proteins, especially difficult-to-express proteins (DtEP) that are insoluble in Escherichia coli, and the target proteins are presented on the surface, for example, in immunologically significant structures. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The structure of the Mycobacterium tuberculosis -derived encapsidation protein is shown as an example of a crystallizable protein.

[0015] Figure 2 An expression vector according to one embodiment of the present invention is shown (T7, T7 promoter; NC, viral nucleocapsid coding site; Enc, envelope protein coding site; Target, target protein coding site; MCS, multiple cloning site).

[0016] Figure 3 An illustrative concept of the present invention is shown.

[0017] Figure 4Shown are fusion protein constructs used to explore the suitability of a combination of a crystallizable protein and a viral nucleocapsid (NC) as fusion partners for the generation of self-assembling nanoparticles.

[0018] Figure 5 and Figure 6 The fusion protein construct regions in the expression vector according to one embodiment of the present invention are respectively shown.

[0019] Figure 7 Shows confirmation Figure 4 SDS-PAGE results of the fusion protein construct expressed in E. coli.

[0020] Figure 8 The transmission electron microscope (TEM) was used to observe Figure 4 Results of the product (self-assembled nanoparticles) obtained by expression of the fusion protein construct.

[0021] Fig. 9 The analysis by size exclusion chromatography (SEC) is shown. Figure 4 Results of the product (self-assembled nanoparticles) obtained by expression of the fusion protein construct.

[0022] Fig.10 Cryo-electron microscopy (Cryo-EM) is shown. Figure 4 Results of the product (self-assembled nanoparticles) obtained by expression of the fusion protein construct.

[0023] Fig.11 The analysis is shown by Figure 4 The expression of the fusion protein constructs results in the location of the N-terminal and C-terminal regions of the fusion protein on the obtained product (self-assembled nanoparticles).

[0024] Fig.12 Shows confirmation Figure 5 SDS-PAGE results of the fusion protein construct expressed in E. coli.

[0025] Fig.13 The TEM observation shows Figure 5 Results of the product (self-assembled nanoparticles) obtained by expression of the fusion protein construct.

[0026] Fig.14 Shows the exploration through Figure 5 The expression of the fusion protein construct results in the obtained product (self-assembled nanoparticles) being a result of the functionality of the antigen.

[0027] Fig.15 Shows confirmation Figure 6SDS-PAGE results of the fusion protein construct expressed in E. coli.

[0028] Fig.16 The TEM observation shows Figure 6 Results of the product (self-assembled nanoparticles) obtained by expression of the fusion protein construct. DETAILED DESCRIPTION

[0029] According to one embodiment of the present invention, an expression vector for producing target protein-fused self-assembling nanoparticles is provided, the expression vector comprising a polynucleotide encoding a crystallizable protein and a viral nucleocapsid as a fusion partner of the target protein.

[0030] The term "target protein" used herein refers to a protein produced in large quantities by a person of ordinary skill in the art, and includes all proteins that can be expressed in a host cell after a polynucleotide encoding the protein is inserted into a recombinant vector.

[0031] 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.

[0032] The term "expression vector" as used herein is a vector for expressing a polynucleotide encoding a polypeptide of interest, which has been introduced into a heterologous or homologous host cell, and refers to a circular or linear polynucleotide, such as a DNA or RNA molecule. The expression vector may include a promoter sequence and / or a terminator sequence, and may also include components required for the vector, such as an origin of replication, a selection marker, a polyadenylation signal, etc.

[0033] 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.

[0034] The expression vector of the present invention can include the polynucleotide encoding the target protein, or can be used to include the polynucleotide encoding the target protein in the future. In particular, in the latter case, as a component that allows the polynucleotide encoding the target protein to be easily included, it is preferred to include a restriction enzyme recognition site commonly referred to as a multiple cloning site (MSC).

[0035] The present invention is based on the following new research results: when a crystallizable protein and a viral nucleocapsid are used as fusion partners of a target protein, the target protein can be effectively expressed (for example, a protein that is insoluble when no fusion partner is used or when another existing fusion partner is used can be soluble); and when a crystallizable protein and a viral nucleocapsid are used as fusion partners of a target protein, nanoparticles in which the target protein self-assembles can be effectively produced.

[0036] Especially, the viral nucleocapsid used as fusion partner in the present invention has the advantage of minimizing interference to target protein, because viral nucleocapsid has very small size (short peptide).When using this fusion partner, there is the advantage of producing the target protein with improved solubility, and usually, the larger the size of the fusion partner, the more helpful in enhancing the water solubility of the target protein.However, the structural interference to adjacent target protein increased due to the size of the fusion partner may also hinder the generation of physiologically active structure.Therefore, if possible, it is very important to find that the size is small, but the solubility and active fusion partner of the target protein are increased.Especially, in the present invention, the viral nucleocapsid used as fusion partner has very small size (short peptide), so it has the advantage of minimizing structural interference to target protein and minimizing structural interference to the self-assembly (in the present invention, self-assembly into nanoparticles) of target protein monomer.Usually, in order to avoid this structural interference, conventional technology needs to remove the fusion partner by treating the fusion protein with a specific cutting enzyme after generation, therefore, the production cost and purification process increased due to the use of expensive enzymes and additional cutting are still obstacles.The present invention can significantly improve this problem technically.

[0037] 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 the zinc affinity purification method, the fusion protein can be easily purified without a separate component for purification, such as a histidine tag.

[0038] In the present invention, the viral nucleocapsid may be a conventionally known viral nucleocapsid, and it may be a fragment or variant of a viral nucleocapsid, as long as the activity of the present invention is retained, in other words, the activity of forming self-assembled nanoparticles is retained, in which the viral nucleocapsid is used together with the crystallizable protein as a fusion partner of the target protein to allow the target protein to be soluble expressed, particularly in Escherichia coli, and to be presented on the surface, particularly in an immunologically significant structure.

[0039] In the present invention, the viral nucleocapsid may be a nucleocapsid of a retrovirus (eg, human immunodeficiency virus-1 (HIV-1), SIV or MuLV).

[0040] In the present invention, the viral nucleocapsid is preferably the nucleocapsid of human immunodeficiency virus (HIV).

[0041] 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 of SEQ ID NO: 1, more preferably amino acids 14, 17, 22, 27, 35, 38, 43, and 48, 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 and satisfies the above sequence identity.

[0042] 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 amino acid 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 preferably includes a nucleotide sequence encoding an amino acid sequence that 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, 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 and satisfies the above sequence identity.

[0043] Sequence identity used herein refers to 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 the amino acid or nucleotide sequence is relevant to the quantity of the same amino acid or nucleotide at each position shared by 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.

[0044] In the present invention, crystallizable proteins can be used without limitation, as long as each protein is a protein having an activity of forming an assembly such as a cage-shaped complex protein as one unit. For example, as the crystallizable protein, encapsulated protein or ferritin can be used.

[0045] In the present invention, conventionally known encapsulating protein or ferritin and fragments of encapsulating protein or ferritin may be used as long as the activity of forming an assembly (eg, a cage-shaped complex protein as a unit) is maintained.

[0046] An encapsulation protein derived from an organism such as Mycobacterium tuberculosis, Thermotoga maritima, Pyrococcus furiosus, or Myxococcus xanthus may be used, and preferably, an encapsulation protein derived from Mycobacterium tuberculosis is used.

[0047] In the present invention, the crystallizable 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.

[0048] In the present invention, the polynucleotide encoding the crystallizable 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 amino acid sequence of SEQ ID NO: 5 or 7, or the nucleotide sequence of SEQ ID NO: 5 or 7.

[0049] The expression vector of the present invention can be used to produce target protein-fused self-assembled nanoparticles in cells selected from all organisms including prokaryotes and eukaryotes, preferably microorganisms of the genus Escherichia, more preferably Escherichia coli.

[0050] In the expression vector of the present invention, the polynucleotide encoding the crystallizable 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, for example 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 crystallizable 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 necessary, the components may be arranged in various ways, or the number of these components may vary.

[0051] In one embodiment, the expression vector of the present invention comprises a polynucleotide encoding a crystallizable protein and a viral nucleocapsid as a fusion partner of a target protein, and the polynucleotide encoding crystallizable protein and the viral nucleocapsid connected to the N-terminal of the crystallizable protein, and comprises a target protein coding site or a cloning site for connecting the target protein to the C-terminal of the crystallizable protein. According to this form, the self-assembling nanoparticle can be produced in a manner such that the target protein is exposed to the outside of the nanoparticle and the viral nucleocapsid is located at the inside of the nanoparticle, which is particularly effective in producing the antigen presenting nanoparticle for immune response. As a specific example of an embodiment, the polynucleotide comprises a construct, wherein a promoter, a viral nucleocapsid coding site, a crystallizable protein coding site, a target protein coding site or a cloning site (e.g., a restriction enzyme recognition site, e.g., MCS) and a terminator for inserting a target protein encoding polynucleotide are operably connected successively in a 5' to 3' direction.

[0052] In another embodiment, the expression vector of the present invention includes a polynucleotide encoding a crystallizable protein and a viral nucleocapsid as a fusion partner of a target protein, and the polynucleotide encodes a crystallizable protein and a viral nucleocapsid connected to the N-terminus of the crystallizable protein, and includes a target protein coding site or a cloning site for connecting the target protein between the crystallizable 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, such as an MCS), a crystallizable protein coding site, and a terminator are operably connected in sequence in a 5' to 3' direction.

[0053] In another embodiment, the expression vector of the present invention includes a polynucleotide encoding a crystallizable protein and a viral nucleocapsid as a fusion partner of a target protein, and the polynucleotide encodes a crystallizable protein and a viral nucleocapsid connected to the N-terminus of the crystallizable protein, and includes a cloning site for connecting the target protein at the C-terminus of the crystallizable protein and between the crystallizable protein and the viral nucleocapsid. In this case, the user can choose to position the target protein at the C-terminus or the N-terminus of the crystallizable protein. As a specific example of an embodiment, the polynucleotide includes a construct, wherein a promoter, a viral nucleocapsid coding site, a first cloning site (e.g., a restriction enzyme recognition site, e.g., MCS) for inserting a polynucleotide encoding a target protein, a crystallizable protein coding site, a second cloning site (e.g., a restriction enzyme recognition site, e.g., MCS) for inserting a polynucleotide encoding a target protein, and a terminator are sequentially operably connected in a 5' to 3' direction.

[0054] The expression vector of the present invention may include a T7 promoter and a T7 terminator to regulate the transcription of the target protein and fusion partner encoding DNA through the bacteriophage T7 system. Accordingly, the expression vector of the present invention can more effectively produce the target protein in Escherichia coli.

[0055] The expression vector of the present invention can be prepared using conventional recombinant methods based on conventional expression vectors, for example.

[0056] According to another embodiment of the present invention, a host cell transformed with an expression vector is provided, wherein the expression vector is used to produce self-assembling nanoparticles fused with the target protein of the present invention.

[0057] The host cell in the present invention can be selected from cells of all organisms including prokaryotes and eukaryotes, preferably cells of microorganisms of the genus Escherichia, more preferably cells of Escherichia coli.

[0058] The term "conversion" or "introduction" as used herein refers to the introduction of a polynucleotide into a host so that the polynucleotide can be replicated as an extrachromosomal element or by completing chromosomal integration. The transformation method using the expression vector according to the present invention can 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.

[0059] According to another embodiment of the present invention, as a method for effectively producing self-assembled nanoparticles of target protein fusion in heterologous or homologous host cells, a method for producing self-assembled nanoparticles of target protein fusion is provided, the method comprising manufacturing an expression vector for producing self-assembled nanoparticles of target protein fusion, the expression vector comprising polynucleotides encoding the target protein and a crystallizable protein and a viral nucleocapsid as fusion partners of the target protein; producing a transformant by introducing the expression vector for producing self-assembled nanoparticles of target protein fusion into a host cell; and culturing the transformant.

[0060] 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 previously described matters.

[0061] 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 cultivating 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, the proliferation of the Escherichia coli containing the expression vector can be preferentially induced by adding the corresponding antibiotic (for example, ampicillin) to the culture medium and cultivating the cells. 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).

[0062] The following examples are provided to help understand the present invention, and the following contents do not limit the scope of the present invention.

[0063] Example

[0064] Preparation Example 1

[0065] To explore the suitability of the combination of crystallizable protein and viral nucleocapsid (NC) as fusion partners for the production of self-assembled nanoparticles, vectors were fabricated to express human immunodeficiency virus (HIV) NC linked to the N-terminus of crystallizable protein, followed by encapsidation protein and six histidine tags, with a tobacco etch virus (TEV) protease cleavage site present between them ( Figure 4 ).

[0066] The vector was manufactured based on the pGEMEX-1 (Promega) vector designed to regulate the expression of the target protein by the T7 promoter, and the sequence of SEQ ID NO: 5 was used as the sequence encoding the encapsidated protein, and the sequence of SEQ ID NO: 2 (or SEQ ID NO: 3) was used as the sequence encoding NC.

[0067] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:8, and the amino acid sequence of the fusion protein expected to be expressed is identical to SEQ ID NO:9.

[0068] Example 1

[0069] The carrier for producing self-assembled nanoparticles of fusion proteins was manufactured in the following form: NC was linked to the N-terminus of the crystallizable protein encapsulating protein, six histidine and TEV protease cleavage sites were present in between, and the target protein as the COVID19 receptor binding domain (RBD) was linked to the C-terminus of the encapsulating protein ( Figure 5 ).

[0070] The manufacturing method was the same as that of Preparation Example 1, but the sequence encoding the NC-6×His tag-TEV protease cleavage site-encapsulation protein-target protein construct was inserted into the MCS of the pGEMEX-1 (Promega) vector.

[0071] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:10, and the amino acid sequence of the fusion protein expected to be expressed is identical to SEQ ID NO:11.

[0072] Example 2

[0073] The carrier for producing self-assembling nanoparticles of fusion proteins was manufactured in the following form: the target protein influenza antigen (H5N1 HA: H5gd) was linked to the N-terminus of the crystallizable protein ferritin, NC was linked to the N-terminus of the influenza antigen, and a six-histidine tag and a TEV protease cleavage site were present between the NC and the influenza antigen ( Figure 6 ).

[0074] The manufacturing method was the same as that of Preparation Example 1, but the sequence encoding the NC-6×His tag-TEV protease cleavage site-target protein-ferritin construct was inserted into the MCS of the pGEMEX-1 (Promega) vector.

[0075] The sequence of the construct region in the manufactured vector is identical to SEQ ID NO:12, and the amino acid sequence of the expected expressed fusion protein is identical to SEQ ID NO:13.

[0076] Experimental Examples

[0077] The vectors of the Preparation Examples and Examples were transformed into E. coli SHuffle / T7 (NEB) or SHuffle / T7 / pLsS (NEB) and cultured. Shuffle / T7 / pLysS was prepared by the following process: pLysS One Shot was prepared from BL21 Star (DE3) using Mini-prep TMpLysS plasmid was extracted from competent Escherichia coli (Invitrogen), and the extracted plasmid was introduced into Shuffle / T7 (NEB) competent Escherichia coli, and selected using chloramphenicol (CM) (antibiotic marker included in pLysS). All transformed Escherichia coli were cultured in LB medium containing a single 50 μg / mL ampicillin or 50 μg / mL ampicillin and 34 μg / mL chloramphenicol. The culture temperature was set to 16°C to 37°C. When the OD600 value of Escherichia coli became 0.5 or higher, IPTG was added at a level of 0 μM to 1 mM to activate the T7 promoter, and after adding IPTG, 3 hours were cultivated at 37°C or 5 hours were cultivated at 25°C to fully produce protein. The fully cultured Escherichia coli was centrifuged to remove the supernatant, and then stored. Subsequently, 0.3 mL of PBS was added to the Escherichia coli harvest corresponding to 5 mL of LB medium, and ultrasonic treatment was performed 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.

[0078] First, as a result of inducing the expression of the fusion protein by introducing the vector of Preparation Example 1 into Escherichia coli, Figure 7 As shown, high levels of fusion protein were expressed normally, i.e., with the expected size and solubility. This suggests that the combination of encapsulated protein and NC has the potential to be used as fusion partners for the generation of self-assembled nanoparticles fused to the target protein.

[0079] Furthermore, as a result of observing the product obtained by expression induction by transmission electron microscope (TEM), as Figure 8 As shown, the product is in the form of nanoparticles.

[0080] Furthermore, as a result of size exclusion chromatography (SEC) of the product obtained by expression induction, e.g. Fig. 9 As shown, the products show a constant size distribution.

[0081] Furthermore, as a result of observing the products obtained by expression induction by Cryo-EM, e.g. Fig.10 As shown, it was shown to have a spherical nanoparticle form. In addition, as a result of analyzing the positions of the N-terminus and the C-terminus of the fusion protein after Cryo-EM, as Fig.11As shown in , the N terminus of the fusion protein is located inside the nanoparticle, and its C terminus is located outside the nanoparticle. These results indicate that when NC is linked to the N terminus of the encapsulated protein and the antigen protein as the target protein is linked to the C terminus, the antigen protein is exposed to the outside of the nanoparticle and acts as an antigen, and NC is located inside the nanoparticle, thereby preventing unnecessary interference with the immune response, and thus highly efficient antigen nanoparticles can be produced.

[0082] First, as a result of inducing the expression of the fusion protein by introducing the vector of Example 1 into Escherichia coli, Fig.12 As shown, high levels of the fusion protein were expressed normally, ie, with the expected size and solubility.

[0083] Furthermore, as a result of observing the product obtained by inducing the expression of the vector of Example 1 by transmission electron microscopy (TEM), as Fig.13 As shown, the product is in the form of nanoparticles.

[0084] In addition, the product obtained by inducing the expression of the vector of Example 1, i.e., the self-assembled nanoparticles, was injected into mice intraperitoneally (IP) or intramuscularly (IM) at a suitable dose, and two weeks later, blood was collected from the orbit to obtain serum, and then ELISA was performed. The injection was performed three times at intervals of two weeks. As a result, Fig.14 As shown, IgG titer is shown. This indicates that according to the method of the present invention, self-assembled nanoparticles can be prepared in which the target protein is fused while maintaining its original structure.

[0085] First, as a result of inducing the expression of the fusion protein by introducing the vector of Example 2 into Escherichia coli, Fig.15 As shown, high levels of the fusion protein were expressed normally, ie, with the expected size and solubility.

[0086] Furthermore, as a result of observing the product obtained by inducing the expression of the vector of Example 2 by transmission electron microscopy (TEM), as Fig.16 As shown, the product is in the form of nanoparticles.

[0087] 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.

[0088] <Sequence Listing>

[0089] SEQ ID NO: 1

[0090] Sequence name: Amino acid sequence of HIV nucleocapsid

[0091] Sequence Type: AA

[0092] Name of Organism: Human Immunodeficiency Virus

[0093] sequence:

[0094] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQ MKDCTERQAN

[0095] SEQ ID NO: 2

[0096] Sequence name: Nucleotide sequence encoding HIV nucleocapsid

[0097] Sequence type: DNA

[0098] Name of Organism: Human Immunodeficiency Virus

[0099] sequence:

[0100] CAGCGGGGAAACTTCAGGAACCAGAGAAAAACTGTGAAGTGCTTCAATTGCGGAAAGGAGGGCCACATCGCTAAGAACTGCCGCGCCCCAGAAAGAAAGGCTGCTGGAGATGCGGCAGAGAGGGCCACCAGATGAAGGACTGCACAGAGAGACAGGCAAAC

[0101] SEQ ID NO:3

[0102] Sequence name: Nucleotide sequence encoding HIV nucleocapsid

[0103] Sequence type: DNA

[0104] Organism Name: Synthetic Construct

[0105] sequence:

[0106] CAGCGTGGTAACTTCCGTAACCAGCGTAAAACCGTTAAATGCTTCAACTGCGGCAAAGAAGGCCACATCGCGAAAAACTGCCGTGCGCCGCGTAAAAAAGGCTGCTGGCGTTGCGGCCGTGAAGGCCACCAGATGAAAGATTGCACCGAACGTCAGGCGAAC

[0107] SEQ ID NO: 4

[0108] Sequence name: Amino acid sequence of the encapsidation protein from Mycobacterium tuberculosis Sequence type: AA

[0109] Name of Organism: Mycobacterium tuberculosis

[0110] sequence:

[0111] NNLYRDLAPVTEAAWAEIELEAARTFKRHIAGRRVVDVSDPGGPVTAAVSTGRLIDVKAPTNGVIAHLRASKPLVRLRVPFTLSRNEIDDVERGSKDSDWEPVKEAAKKLAFVEDRTIFEGYSAASIEGIRS ASSNPALLTLPEDPREIPDVISQALSELRLAGVDGPYSVLLSADVYTKVSETSDHGYPIREHLNRLVDGDIIWAPAIDGAFVLTTRGGDFDLQLGTDVAIGYASHDTDTVRLYLQETLTFLCYTAEASVALSH

[0112] SEQ ID NO:5

[0113] Sequence name: Nucleotide sequence encoding the encapsidation protein from Mycobacterium tuberculosis Sequence type: DNA

[0114] Name of Organism: Mycobacterium tuberculosis

[0115] sequence:

[0116] AATAACCTGTACCGGGACCTTGCACCAGTCACTGAGGCTGCGTGGGCCGAGATCGAGCTTGAGGCCGCACGTACGTTTAAGCGTCATATTGCGGGCCGTCGGGTTGTGGATGTATCAGATCCTGGAGGTCCTGTAACCGCTGCGGTATCGACAGGGCGTTTGATTGACGTAAAGGCTCCAACCAACGGTGTCATTGCCCACTTACGCGCCAGCAAGCCTTTAGTTCGTTTAAGAGTGCCCTTTACGTTAAGCCGGAACGAAATTGATGATGTGGAACGTGGGTCAAAGGACAGTGATTGGGAACCAGTGAAAGAGGCGGCGAAGAAGTTGGCCTTCGTCGAAGATCGTACCATATTTGAAGGCTACTCCGCTGCTTCCATAGAAGGGATTCGGTCCGCCTCCTCAAATCCAGCATTAACGCTGCCTGAAGACCCGCGCGAGATTCCTGATGTAATCTCACAGGCACTTTCGGAATTACGTTTGGCAGGGGTCGATGGACCATACAGTGTTCTTTTGAGTGCAGATGTATACACCAAGGTGAGCGAGACCAGTGATCACGGCTATCCAATACGGGAACACCTGAACAGACTTGTCGATGGCGACATAATCTGGGCGCCCGCAATTGACGGCGCATTCGTGTTGACCACCCGTGGTGGTGATTTTGATTTACAATTAGGCACCGATGTTGCGATTGGGTATGCGAGTCATGATACGGACACTGTACGTTTGTATCTTCAAGAGACGCTGACGTTCTTATGTTACACCGCTGAGGCGTCCGTGGCGCTTTCGCAT

[0117] SEQ ID NO:6

[0118] Sequence Name: Amino Acid Sequence of Ferritin from Escherichia coli

[0119] Sequence Type: AA

[0120] Organism Name: Escherichia coli

[0121] Sequence:

[0122] KGDTKVINYLNKLLGNELVAINQYFLHARMFKNWGLKRLNDVEYHESIDEMKHADRYIERILFLEGLPNLQDLGKLNIGEDVEEMLRSDLALELDGAKNLREAIGYADSVHDYVSRDMMIEILRDEEGHIDWLETELDLIQKMGLQNYLQAQIREEG

[0123] SEQ ID NO:7

[0124] Sequence name: Nucleotide sequence encoding ferritin from Escherichia coli

[0125] Sequence type: DNA

[0126] Organism Name: Escherichia coli

[0127] sequence:

[0128] AAAGGTGATACTAAAGTTATAAATTATCTCAACAAACTGTTGGGAAATGAGCTTGTCGCAATCAATCAGTACTTTCTCCATGCCCGAATGTTTAAAAACTGGGGTCTCAAACGTCTCAATGATGTGGAGTATCATGAATCCATTGATGAGATGAAACACGCCGATCGTTATATTGAGCGCATTCTTTTTCTGGAAGGTCTTCCAAACTTACAGGACCTGGGCAAACTGAACATTG GTGAAGATGTTGAGGAAATGCTGCGTTCTGATCTGGCACTTGAGCTGGATGGCGCGAAGAATTTGGCGTGAGGCAATTGGTTATGCCGATAGCGTTCATGATTACGTCAGCCGCGATATGATGATAGAAATTTTGCGTGATGAAGAAGGCCATATCGACTGGCTGGAAACGGAACTTGATCTGATTCAGAAGATGGGCCTGCAAAATTATCTGCAAGCACAGATCCGCGAAGAAGGT

[0129] SEQ ID NO:8

[0130] Sequence name: Nucleotide sequence of fusion protein construct

[0131] Sequence type: DNA

[0132] Organism Name: Synthetic Construct

[0133] sequence:

[0134]

[0135] SEQ ID NO:9

[0136] Sequence name: amino acid sequence of the expected fusion protein

[0137] Sequence Type: AA

[0138] Organism Name: Synthetic Construct

[0139] sequence:

[0140] MQRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANHHHHHHENLYFQGGSNNLYRDLAPVTEAAWAEIELEAARTFKRHIAGRRVVDVSDPGGPVTAAVSTGRLIDVKAPTNGVIAHLRASKPLVRLRVPFTLSRNEIDDVERGSKDSDW EPVKEAAKKLAFVEDRTIFEGYSAASIEGIRSASSNPALTLPEDPREIPDVISQALSELRLAGVDGPYSVLLSADVYTKVSETSDHGYPIREHLNRLVDGDIIWAPAIDGAFVLTTRGGDFDLQLGTDVAIGYASHDTDTVRLYLQETLTFLCYTAEASVALSHDIVDKL

[0141] SEQ ID NO:10

[0142] Sequence name: Nucleotide sequence of fusion protein construct

[0143] Sequence type: DNA

[0144] Organism Name: Synthetic Construct

[0145] sequence:

[0146]

[0147] SEQ ID NO: 11

[0148] Sequence name: amino acid sequence of the expected fusion protein

[0149] Sequence Type: AA

[0150] Organism Name: Synthetic Construct

[0151] sequence:

[0152] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANENLYFQGHHHHHHDDDDGTGSNNLYRDLAPVTEAAWAEIELEAARTFKRHIAGRRVVDVSDPGGPVTAAVSTGRLIDVKAP TNGVIAHLRASKPLVRLRVPFTLSRNEIDDVERGSKDSDWEPVKEAAKKLAFVEDRTIFEGYSAASIEGIRSASSNPALTLPEDPREIPDVISQALSELRLAGVDGPYSVLLSADVYTKVSETSDHGYPIREHLN RLVDGDIIWAPAIDGAFVLTTRGGDFDLQLGTDVAIGYASHDTDTVRLYLQETLTFLCYTAEASVALSHDIVDNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCF TNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLRRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFENKSAPATVCGP

[0153] SEQ ID NO:12

[0154] Sequence name: Nucleotide sequence of fusion protein construct

[0155] Sequence type: DNA

[0156] Organism Name: Synthetic Construct

[0157] sequence:

[0158]

[0159] SEQ ID NO:13

[0160] Sequence name: amino acid sequence of the expected fusion protein

[0161] Sequence Type: AA

[0162] Organism Name: Synthetic Construct

[0163] sequence:

[0164] QRGNFRNQRKTVKCFNCGKEGHIAKNCRAPRKKGCWRCGREGHQMKDCTERQANHHHHHHENLYFQGGTGSGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPTND LCYPGSFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYLGSPSFFRNVVWLIKKNSTYPTIKKSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISIGT STLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSAIMKSELDIVDMKGDTKVINYLNKLLGNELVAINQYFLHARMFKNWGLKRLNDV EYHESIDEMKHADRYIERILFLEGLPNLQDLGKLNIGEDVEEMLRSDLALELDGAKNLREAIGYADSVHDYVSRDMMIEILRDEEGHIDWLETELDLIQKMGLQNYLQAQIREEG

Claims

1. An expression vector for producing target protein-fused self-assembling nanoparticles, the expression vector comprising a polynucleotide encoding a crystallizable protein and a viral nucleocapsid as a fusion partner of the target protein.

2. The expression vector according to claim 1, wherein the target protein is at least one selected from the group consisting of antigens, antibodies, cell receptors, enzymes, structural proteins, serum and cell proteins.

3. The expression vector according to claim 1, wherein the crystallizable protein is encapsidin or ferritin. The expression vector according to claim 1 , wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus (HIV).

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 target protein-fused self-assembled nanoparticles in Escherichia coli.

7. The expression vector according to claim 1, wherein the polynucleotide encodes a crystallizable protein and a viral nucleocapsid linked to the N-terminus of the crystallizable protein, and comprises a cloning site for linking a target protein encoding site or a target protein to the C-terminus of the crystallizable protein.

8. A host cell transformed with the expression vector for producing target protein-fused self-assembled nanoparticles according to any one of claims 1 to 7.

9. A method for producing self-assembled nanoparticles fused to a target protein, the method comprising: Manufacturing an expression vector for producing target protein-fused self-assembling nanoparticles, the expression vector comprising a polynucleotide encoding the target protein and a crystallizable protein and a viral nucleocapsid as a fusion partner of the target protein; Producing a transformant by introducing the expression vector for producing target protein-fused self-assembling nanoparticles into a host cell; as well as The transformants were cultured.

10. The method according to claim 9, wherein the target protein is at least one selected from the group consisting of an antigen, an antibody, a cell receptor, an enzyme, a structural protein, a serum, and a cell protein.

11. The method of claim 9, wherein the crystallizable protein is encapsidin or ferritin.

12. The method of claim 9, wherein the viral nucleocapsid is a nucleocapsid of human immunodeficiency virus (HIV).

13. The method of claim 9, wherein the viral nucleocapsid comprises the amino acid sequence of SEQ ID NO:

1.

14. The method of claim 9, wherein the host cell is Escherichia coli.

15. The method of claim 9, wherein the polynucleotide encodes the crystallizable protein, a viral nucleocapsid linked to the N-terminus of the crystallizable protein, and a target protein linked to the C-terminus of the crystallizable protein.

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