KR102264535B1 - Recombinant expression vector for production of encapsulin-based vaccine and method for manufacturing the same - Google Patents

Abstract

The present invention relates to a fusion protein comprising a polynucleotide encoding an RNA interacting domain (RID) protein, which is a water-soluble enhancement fusion partner for promoting high-efficiency water-soluble expression of encapsulin monomer-target protein fusions in which the target protein is exposed on the surface. More specifically, the present invention provides a recombinant expression vector for producing a high-efficiency vaccine of a target protein using an encapsulin protein, and a method for preparing the same.

Description

Recombinant expression vector for the production of encapsulin-based vaccines and a method for preparing the same {RECOMBINANT EXPRESSION VECTOR FOR PRODUCTION OF ENCAPSULIN-BASED VACCINE AND METHOD FOR MANUFACTURING THE SAME}

The present invention relates to an encapsulin protein and a fusion protein comprising the same for enhancing the expression efficiency of the target protein, and more particularly, a polynucleotide encoding the target protein, the encapsulin protein, and an RNA interacting domain (RID) protein To provide a recombinant expression vector for vaccine production and a method for producing the same, capable of producing a water-soluble vaccine with high efficiency, including a large-sized target protein.

The development of new preventive vaccines for efficient response to rapidly increasing infectious diseases and the development of therapeutic vaccines that help the body fight against diseases (viruses, cancer, etc.) that the body already has are being actively developed. Prophylactic Vaccines are vaccines that induce an immune response by injecting pathogens to prevent disease in healthy general population. Therapeutic Vaccines treat disease in patients with the disease. It is a vaccine that strengthens the immune system (self-antigen injection, etc.) for

Traditional vaccines have been manufactured using an attenuated strain whose toxicity is weakened through long-term subculture based on the process of culturing the pathogen itself or a strain inactivated through compound treatment. However, in this case, the development and manufacturing period is too long, and there is a disadvantage that it cannot be applied to the development of a vaccine against a pathogen that is difficult to culture.

In order to produce a more effective vaccine without being affected by the culture of the pathogen itself, a subunit vaccine using only a specific site protein or a denatured toxin as an antigen among pathogen components has been developed. In this case, it has the advantage of securing higher safety, but also has the disadvantage that multiple administrations and a separate adjuvant are required due to the relatively low immunogenicity.

For the rapid development of a safer and more effective vaccine against the rapidly increasing number of new strains of virus infection, a virus-like particle (VLP) vaccine manufacturing method was recently developed. It is spotlighted as a next-generation vaccine manufacturing technology that can overcome the limitations of existing vaccine production technologies.

Virus-like conformation (VLP) is a highly complex and sophisticated structure in which a viral structural protein is specifically expressed to exhibit a structure similar in appearance to that of a wild-type virus. It has a size of 20 to 100 nm in diameter and can be produced through polymer formation (virion assembly) after specifically expressing viral structural proteins with known sequences using various types of recombinant protein expression systems.

Although it has the same structure and appearance as the actual infectious virus, propagation is impossible because there is no virus-derived gene, but high immunogenicity is induced through high-density & highly ordered conformation. Because its structure is similar to that of wild-type virus, it has the advantage of being able to stimulate both T-cell and B-cell immune pathways in the body. In addition, since there is no genetic material in the formed structure, there is no infection ability, so it has high safety and excellent structural stability. However, since the structure is complex and shows greatly different characteristics for each virus, there are only a few cases of VLPs licensed as commercial vaccines due to the need for a complex manufacturing process or low productivity.

For the production of more improved VLP-based vaccines, a fusion virus-like three-dimensional (chimeric VLP) manufacturing technology in which an external pathogen antigen is introduced into a VLP structure that forms a polymer structure well has been developed and has been utilized in the development of vaccines targeting various pathogens. . This is in the form of expressing a foreign pathogen antigen on the surface of a VLP-based structure composed of structural proteins of other specific viruses or self-assembling proteins. In order to introduce a foreign antigen into the VLP-based structure, a chemical conjugation or genetic fusion method is used. The chemical conjugation method involves chemical fusion of a separately prepared foreign antigen to a previously prepared VLP-based structure through covalent bonding. It is difficult to align antigens in a certain direction, and the density of expressed antigens is relatively low. Disadvantages are pointed out. On the other hand, the genetic fusion method has the advantage that it is possible to manufacture a chimeric VLP in a simpler process by fusion expression of the VLP structural protein gene and the target antigen protein gene at the DNA level. have. However, in this case, only a very small external antigen composed of 10 to 20 amino acids can be introduced. If it is larger than that, the polymer formation by the structural protein is inhibited due to steric hindrance due to structural limitations. do. In particular, when expressed in E. coli for high-efficiency and rapid production, proper folding is not performed, forming an insoluble inclusion body, and VLP production becomes impossible.

In a previous study, the present inventors have revealed that when an RNA interaction domain (RID) including an aminoacyl RNA synthetase N-terminal domain (ARSNTD) is used as a binding partner, it is involved in protein folding and water solubility of various proteins.

In the present invention, in order to more efficiently and stably express a target antigen protein of a large size that was previously impossible to a VLP structure made through self-assembly of encapsulin protein, the RNA-interaction domain ( RID) as a novel fusion partner, it became possible to express the fusion protein of the encapsulin and the target antigen with high efficiency and water solubility. The present invention was completed after confirming that the fusion protein enables the rapid production of a large amount of fusion virus-like stereotactic (chimeric VLP) through self-assembly after purification without the need for refolding.

An object of the present invention is to provide a vector capable of expressing a large-sized target protein more efficiently and stably, and capable of high-efficiency, water-soluble expression of a fusion protein of an encapsulin protein and a target protein, rotavirus or cervical cancer To provide a fusion protein that can be used for prevention or treatment of enteritis or cervical cancer by using a viral antigen protein such as a virus as a target protein.

In order to solve the above problems, the present invention provides a recombinant expression vector for vaccine production comprising an encapsulin protein and a polynucleotide encoding a target protein.

The present invention also provides a recombinant expression vector for vaccine production, comprising a polynucleotide encoding a target protein, an encapsulin protein, and an RNA interacting domain (RID) protein.

According to an embodiment of the present invention, the polynucleotide may further encode a TEV protein for cleavage of the RID.

The present invention also provides a fusion protein comprising a target protein, an encapsulin protein, and an RNA interacting domain (RID) protein.

According to an embodiment of the present invention, the target protein may be linked to the C-terminus of the encapsulin protein.

The present invention also relates to: a) a protein of interest; encapsulin protein; and RID protein; producing a recombinant expression vector for virus vaccine production comprising a polynucleotide encoding, b) introducing the expression vector into a host cell to produce a transformant, and c) the transformant Inducing expression of the recombinant fusion protein by culturing, and provides a vaccine production method comprising the step of obtaining the same.

According to an embodiment of the present invention, the RID protein can be used as a fusion partner to increase the expression level or water solubility of the produced vaccine.

According to another embodiment of the present invention, the RID protein may include the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7 or a partial sequence thereof.

According to another embodiment of the present invention, there is provided a host cell transformed with the expression vector, and in the present invention, E. coli may be used as the host cell.

According to one embodiment of the present invention, the encapsulin protein may include the amino acid sequence of SEQ ID NO: 1 or a partial sequence thereof.

According to another embodiment of the present invention, the target protein may be one or more selected from the group consisting of viral antigen proteins, antibodies, cell receptors, enzymes, structural proteins, serum and cellular proteins.

According to another embodiment of the present invention, the viral antigen protein may be a cervical cancer virus antigen protein or a rotavirus antigen protein.

The fusion protein prepared according to the present invention has the advantage of being able to more efficiently and stably express a large-sized target protein.

According to an embodiment of the present invention, there is an advantage in that the fusion protein of the encapsulin and the target protein including RID as a novel fusion partner can be expressed in a water-soluble manner with high efficiency.

The fusion protein prepared according to the present invention has a structure similar to that of the wild-type virus, and thus can stimulate both T-cell and B-cell immune pathways in the body, thereby having a large immune effect.

The fusion protein prepared according to the present invention can use a target protein as a rotavirus antigen protein, and can prevent or treat enteritis using the fusion protein.

The fusion protein prepared according to the present invention can use the target protein as a cervical cancer virus antigen protein, and can prevent or treat cervical cancer by using it.

According to the vaccine production method of the present invention, it is possible to rapidly produce a large amount of fusion protein to shorten the vaccine production time.

1 is a schematic diagram of a recombinant expression vector for producing Encapsulin protein according to an embodiment of the present invention and a result of high-efficiency water-soluble protein expression. 1a is a protein structure of encapsulin predicted using PDB. 1B is a schematic diagram showing the structure of a pET9a-Encapsulin recombinant expression vector. Figure 1c is the result of confirming the high-efficiency water-soluble expression of the encapsulin protein using the expression vector by SDS-PAGE.
Figure 2a is a chromatogram result when the Encapsulin-Linker protein expressed according to an embodiment of the present invention is purified through nickel affinity chromatography, Figure 2b is the result of confirming the separation pattern of the Encapsulin-Linker protein by SDS-PAGE to be. Figure 2c is a chromatogram result when the encapsulin protein eluted from nickel affinity chromatography is purified through ion resin exchange chromatography, Figure 2d is the encapsulin protein eluted from nickel affinity chromatography ( Encapsulin) protein purification was confirmed by SDS-PAGE.
Figure 3a is an elution graph after the purified encapsulin (Encapsulin) protein is subjected to size exclusion chromatography. Figure 3b is the result of confirming the separation pattern of the encapsulin (Encapsulin) protein by SDS-PAGE after performing size exclusion chromatography. 3c and 3d are the results of analyzing the diameter distribution of the purified encapsulin protein three-dimensional through DLS (Dynamic Light Scattering). Figure 3e is a result of confirming the overall appearance of purified encapsulin (Encapsulin)-based VLP (Virus-like particle) through a transmission electron microscope (TEM).
Figure 4a is an enzyme cleavage site and ligation site of an appropriate sequence with RID, a fusion partner for enhancing expression level and water solubility, in order to prepare an encapsulin-based virus-like three-dimensional structure expressing rotavirus VP8* according to an embodiment of the present invention; is applied to the Encapsulin RV VP8* fusion protein to illustrate the expression strategy. Figure 4b shows the structure of the expression vector corresponding to Figure 4a, pET9a-MSAVKAA-RID-Linker-TEV-Linker-Encapsulin into which additional sequences for expression level and water solubility enhancement fusion partner MSAVKAA-RID and TEV clearance efficiency are inserted. Corresponds to the vector structure of -Linker-Rotavirus VP8*. 4c is an SDS-PAGE result showing the effect of a fusion protein of an encapsulin protein and an antigen protein on the fusion expression when RID, a fusion partner for enhancing expression and water solubility of the peptide of the present invention is applied. Changes in relative expression levels for Encapsulin and Encapsulin-Linker-VP8* can be confirmed, and Encapsulin-Linker is a recombinant protein (size 32.1 kDa) and rotavirus ( Rotavirus, RV) antigen, VP8* is the expression result of Encapsulin-Linker-VP8* recombinant protein (size 50.3 kDa) bound to the C-terminus. In the case of Figure 4d, the TEV cleavage site applied between RID and encapsulin according to an embodiment of the present invention was treated with TEV enzyme, and the separation aspect and purity of the protein during ion exchange resin purification through SDS-PAGE This is the confirmed result.
Figure 5a is a result of analyzing the overall diameter distribution of the VLP formed by the purified encapsulin (Encapsulin) protein and the antigen protein fusion protein through DLS (Dynamic Light Scattering). 5B is a result of confirming the overall appearance of an encapsulin-based virus-like particle (VLP) having a target protein expressed on the surface through a transmission electron microscope (TEM).
6a is a schedule from inoculation of a sample to a mouse according to an embodiment of the present invention until serum acquisition, and a table of composition and content of a sample injected into a mouse according to an embodiment of the present invention.
Figure 6b is an enzyme-linked immunosorbent assay (ELISA (enzyme-linked immunosorbent assay)) to measure the target protein-specific antibody response in the serum obtained from the animal test group inoculated with the VLP formed by the fusion protein of the encapsulin and the target protein. It is the result of analysis.
Figure 6c shows the HBGA Binding Assay components and analysis process in order to measure the ability of the target protein to bind to the receptor HBGA, Figure 6d is the result of measuring the binding between the target protein and the receptor (Led (H type 1)) to be.
Figure 6e is a HBGA Blocking Assay component for measuring the ability of the target protein-specific antibody in the serum of the animal test group inoculated with the VLP formed by the fusion protein of the encapsulin and the target protein to inhibit the binding between the target protein and the receptor; The analysis process is shown and FIG. 6f is a result of measuring the degree to which the binding between the target protein and the receptor (Led (H type 1)) is inhibited by the immune serum.
7A is a schematic diagram of a polynucleotide encoding E7 LP, which is a peptide having an amino acid sequence of a human papillomavirus, and an encapsulin protein, and a vaccine prepared by the polynucleotide according to an embodiment of the present invention. Figure 7b shows the structure of the expression vector corresponding to Figure 7a, pET9a-MSAVKAA-RID-Linker-TEV-Linker-Encapsulin into which additional sequences for expression level and water solubility enhancement fusion partner MSAVKAA-RID and TEV clearance efficiency are inserted. This is an image of the vector structure of -Linker-Rotavirus E7 LP. The effect on the fusion expression of the fusion protein of the encapsulin protein and the target protein when RID, which is a fusion partner for enhancing expression and water solubility, was applied was confirmed through the results of FIGS. 7c is an SDS-PAGE result of Encapsulin-Linker-E7 recombinant protein (size 36.1 kDa) in which E7 LP, an antigen of Human papillomavirus (HPV) Type 16, is bound to the C-terminus. Figure 7d is the result of confirming the change in water solubility and expression level of the protein after adding RID to the N-terminus of Encapsulin-Linker-HPV E7 LP by SDS-PAGE. Through this, the necessity of RID, a fusion partner, was confirmed even when the human papillomavirus antigen was added to the C-terminus like rotavirus.
8a is a chromatogram result when Encapsulin-Linker-HPV E7 LP protein expressed according to an embodiment of the present invention is purified through nickel affinity chromatography, and FIG. 8b is isolation of Encapsulin-Linker-HPV E7 LP protein. It is the result of confirming the aspect by SDS-PAGE. Figure 8c is a chromatogram result when the Encapsulin-Linker-HPV E7 LP protein fusion is purified by ion resin exchange chromatography in nickel affinity chromatography, and Figure 8d is the purification aspect of Encapsulin-Linker-HPV E7 LP protein. This is the result confirmed by SDS-PAGE.
9a is an SDS-PAGE result confirming the purification aspect and purity of the fusion protein in the purification process from the protein expression of Encapsulin-Linker-HPV E7 LP to TEV enzyme treatment and ion exchange resin chromatography in the present invention. Figure 9b shows the results of analysis through DLS (Dynamic Light Scattering) to confirm the overall diameter distribution of the VLP of the purified encapsulin protein-target protein fusion. Figure 9c is a result of confirming the overall appearance of the VLP of the encapsulin (Encapsulin)-based fusion protein on which the target protein is expressed on the surface through a purified transmission electron microscope (TEM).

Hereinafter, preferred embodiments of the present invention will be described in detail. Advantages and features of the present invention, as well as embodiments for achieving them, will become apparent with reference to the following embodiments. However, the present invention is not limited to the embodiments published below, but may be implemented in various different forms, and only these embodiments allow the publication of the present invention to be complete, and common knowledge in the art to which the present invention pertains. It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims. Like reference numerals refer to like elements throughout.

Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used with the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not to be interpreted ideally or excessively unless clearly defined in particular. The terminology used herein is for the purpose of describing the embodiments and is not intended to limit the present invention. As used herein, the singular also includes the plural unless specifically stated otherwise in the phrase.

The present invention provides a recombinant expression vector for vaccine production, comprising a polynucleotide encoding an encapsulin protein and a target protein.

As used herein, "encapsulin protein" may be fused to a target protein and expressed together to enhance the expression efficiency of the protein. According to an embodiment of the present invention, the encapsulin protein comprises the amino acid sequence of SEQ ID NO: 1 or a partial sequence thereof.

The encapsulin protein used in the present invention may be obtained from Thermotoga maritima , Pyrococcus furiosus , Myxococcus xanthus and the like, and most preferably from Thermotoga maritima. When using the encapsulin protein obtained from Thermotoga maritima , it is more advantageous for use as a vaccine because it can form small-sized particles.

About 60 encapsulin proteins of the present invention can be self-assembled to form particles having a diameter of 30 to 32 nm. This has the advantage of being able to maintain a stable form even with changes in pH and temperature. Therefore, various cargo proteins or substances can be loaded and various antigens can be attached to the exposed surface domain (preferably C-terminal). The recombinant expression vector prepared according to an embodiment of the present invention can be expressed and self-assembled in various yeasts or cultured cells other than bacteria by using an encapsulin protein.

As used herein, the term “target protein” refers to a protein that a person skilled in the art wants to mass-produce, and which can be expressed in a host cell by inserting a polynucleotide encoding the protein into a recombinant expression vector.

According to an embodiment of the present invention, the target protein includes a viral antigen protein, an antibody, a cell receptor, an enzyme, a structural protein, a serum and a cellular protein. According to a preferred embodiment of the present invention, the viral antigen protein comprises the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, or a partial sequence thereof.

According to the present invention, the viral antigen protein corresponding to the target protein includes, but is not limited to, a rotavirus antigen protein and a cervical cancer virus antigen protein. For example, it may be an antigen protein having a size of 10 to 300 amino acids, preferably a viral antigen protein having a size of 20 to 25 amino acids.

As used herein, the term "expression vector" is a linear or circular DNA molecule composed of a fragment encoding a target (target) protein operably linked to additional fragments that serve for transcription of the expression vector. Such additional fragments include promoter and terminator sequences. Expression vectors also include one or more replication origins, one or more selectable markers, and the like. Expression vectors are generally derived from plasmid or viral DNA, or contain elements of both. As used herein, the term “operably linked” refers to the arrangement of fragments in a promoter to act to initiate transcription and progress through the coding sequence to the termination code.

In the expression vector according to the present invention, the expression vector may be a plasmid, a viral vector, a phage particle or a genome insert. After the expression vector is transformed into a host cell, it can be cloned independently of the genome of the host cell or integrated into the genome of the host cell.

The present invention also provides a recombinant expression vector for vaccine production, comprising a polynucleotide encoding a target protein, an encapsulin protein, and an RNA interacting domain (RID) protein. According to an embodiment of the present invention, the polynucleotide may further encode a "linker", and the linker may include the gene sequence of SEQ ID NO: 8, 9 or 10.

As used herein, the term “RID”, “RNA interacting domain”, “N terminal appended RNA binding domain of Lysyl tRNA synthetase”, or “LysRS RNA interacting domain” refers to LysRS RNA and other proteins It refers to a unique N-terminal extension site involved in the interaction between the liver.

According to an embodiment of the present invention, the RID protein may be used as a fusion partner to increase the expression level or water solubility of the vaccine.

According to a preferred embodiment of the present invention, the RID protein may include a domain for increasing the expression level of the vaccine (hereinafter, EE domain) and a domain for increasing water solubility (hereinafter, SE domain). For example, the EE domain may include the amino acid sequence of SEQ ID NO: 5 or a partial sequence thereof, and the SE domain may include the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or a partial sequence thereof.

In the recombinant expression vector for vaccine production of the present invention, the proteolytic enzyme may be TEV.

According to a preferred embodiment of the present invention, the polynucleotide encoding the target protein may encode the TEV protein for RID cleavage.

The present invention also provides a host cell transformed with the expression vector.

As used herein, the term “transformation” or “introduction” refers to the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or by chromosomal integration completion. The method for transforming the expression vector according to the present invention is electroporation, calcium phosphate (CaPO4) method, calcium chloride (CaCl2) method, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method. , a cationic liposome method or lithium acetate-DMSO method, but is not limited thereto.

In the host cell according to the present invention, the host cell is preferably a host cell having high DNA introduction efficiency and high DNA expression efficiency, and all microorganisms including prokaryotic and eukaryotic microorganisms may be used. Preferably, the host cell may be E. coli.

The present invention also provides a fusion protein comprising a target protein and an encapsulin protein, and the fusion protein may further include an RNA interacting domain (RID) protein.

As used herein, the term “fusion protein” refers to a protein in which another protein is linked to the N-terminus or C-terminus of a target protein sequence or a different amino acid sequence is added. According to a preferred embodiment of the present invention, the target protein may be linked to the C-terminal of the encapsulin protein. In addition, in the present invention, the fusion protein may be a vaccine produced by the recombinant expression vector of the present invention.

According to an embodiment of the present invention, a fusion protein may be formed using the recombinant expression vector, and the fusion protein may be used as a vaccine by itself or through any additional process.

The encapsulin protein according to the present invention can improve the expression efficiency of the target protein. Also, the fusion protein in which the encapsulin protein is coupled to RID, which is well known as a water-soluble enhancing protein, is not only the expression efficiency of the target protein but also the water-soluble By improving it, it can be usefully used in the production of fusion proteins.

According to another embodiment of the present invention, a) a target protein; and encapsulin protein; producing a recombinant expression vector for virus vaccine production comprising a polynucleotide encoding, b) introducing the expression vector into a host cell to produce a transformant, and c) the transformation There is provided a method for producing a vaccine comprising culturing a sieve to induce expression of the recombinant fusion protein, and obtaining the same.

Hereinafter, the present invention will be described in more detail through examples.

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

[Example 1]

Preparation of fusion protein containing encapsulin

<Example 1-1>

Encapsulin (Thermotoga maritima MSB8 (Sequence ID: NC_000853, Protein ID: WP_004080898.1)) gene sequence from Thermotoga Maritama having the protein structure of FIG. 1A was used for high-efficiency production of a fusion protein containing encapsulin. (SEQ ID NO: 1). Based on the above gene sequence, a gene sequence synthesized after codon optimization in E. coli was prepared (SEQ ID NO: 1). (The gene sequence of Example 1-1 was prepared by requesting Bionics to synthesize the Encapsulin gene.) Then, for the purpose of purifying Encapsulin, the linking site of the sequence as shown in SEQ ID NO: 10 was inserted at the C-terminus did. (Amino acid sequence GGGSGGGSHHHHHHGGGS (SEQ ID NO: 10), (HHHHHH: amino acid corresponding to 6xHIS tag)) The prepared complex is named “Enc”. The gene of the above sequence was inserted using NdeI and BamHI, which are cleavage enzymes present in the MCS of the pET-9a (Novagene, 69431-3CN) (SEQ ID NO: 4) expression vector.

<Example 1-2>

The expression vector prepared in Example 1-1 was transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed E. coli were cultured in 3 ml of LB medium containing 50 μg/ml kanamycin at 37° C. at 250 rpm for 5-7 hours, then 1 ml was transferred to 10 ml, diluted 10 times, and further cultured. After about 2-4 hours, when O.D600 0.8~0.9 was reached, 0.4mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added and 17-19 hours at 16°C ( up to 21 hours). The culture medium obtained only the precipitated E. coli through centrifugation and stored frozen at -80°C cryogenically. 0.3 ml of lysis buffer solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 10 mM imidazole) was added to the E. coli harvest corresponding to 3 ml of culture medium and sonicated After grinding, the whole lysate (T, Total lysate) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) through centrifugation, and then the separated protein was analyzed by SDS-PAGE.

1 is a schematic diagram of a recombinant expression vector for producing Encapsulin protein according to Example 1-1 and a result of high-efficiency water-soluble protein expression. 1a is a predicted protein structure of encapsulin using PDB, and FIG. 1b is a schematic diagram showing the structure of a pET9a-Encapsulin recombinant expression vector. As shown in FIG. 1c , it was confirmed by SDS-PAGE whether high-efficiency water-soluble expression of the encapsulin protein was used using the expression vector. The protein expressed according to Examples 1-1 and 1-2 was confirmed to be soluble in E. coli with a size of 32.1 kDa.

<Example 1-3>

Cells obtained by culturing and inducing expression of 500 ml of E. coli using the culture method in the same manner as in Example 1-2 were mixed with 75 ml of lysis buffer [A buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl) , 5% glycerol, 0.1% Triton X-100 and 10 mM imidazole]. The cell resuspension was disrupted using an ultrasonic cell disrupter (sonication: pulse 3s/17s, 35% amplitude, 1min 30s, 4times total). The lysed cells were centrifuged at 13500 rpm at 4° C. for 10 minutes, and the supernatant was collected and Ni+-affinity chromatography was performed using AKTA (GE healthcare). First, the cation-exchange resin column HisTrap HP (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK, 5mL size) column was heated with buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.2 % Tween-20 and 10 mM imidazole), and then the aqueous solution containing the water-soluble protein was loaded onto the equilibrated column at a flow rate of 1 ml/min. Purification of encapsulin protein was performed using buffer A and buffer B containing 1M imidazole (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.2% Tween-20 and 1M imidazole). Imidazole was carried out in the form of increasing injection in a linear concentration gradient in the concentration range of 10mM to 1M using this, and through this, fractions of 2ml each were obtained.

As shown in Figures 2a and 2b, the eluate was subjected to SDS-PAGE to confirm the separation of encapsulin protein, and then the eluate of the fraction was treated with a dialysis buffer (50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 0.2% Tween-20) at 9° C. for 18 hours.

After the first purification and dialysis, a biochemical analysis was performed to determine whether the encapsulin protein forms a VLP, and chromatography (Ion Exchange Chromatography) was performed. Encapsulin proteins obtained by the dialysis were purified by ion exchange resin chromatography using an FPLC system (AKTA, GE healthcare). Column (HiTrap Q FF, 1 mL size (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK)) is buffer A (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 5% glycerol, 0.2% Tween-20) was equilibrated with and loaded at a flow rate of 1 ml / min Encapsulin protein was eluted by injecting NaCl having a linear concentration gradient of 10 mM to 1 M, buffer solution A and buffer solution B (50 mM Tris-HCl) (pH 7.5), 1M NaCl, 5% glycerol, 0.2% Tween-20) was purified through a linear concentration gradient, and fractions of 1 ml each were obtained.

A fusion protein in which other proteins derived from E. coli were purified through ion exchange resin chromatography using a linear gradient (10 mM → 1M NaCl) was obtained ( FIGS. 2C and 2D ). Purified encapsulin (Encapsulin) protein was stored at 4 ℃ in a state containing 20% glycerol (glycerol) ratio.

Additionally, purification using size exclusion chromatography (SEC) was performed. Analysis was performed using an FPLC system (AKTAPurifier, GE Healthcare) (FIGS. 3a and 3b). The prepared sample was equilibrated with a buffer solution (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 5% glycerol) under the same conditions as the dialysis solution on a gel-filtration column (Superose™ 6 Increase 10/300 GL (GE Healthcare Life) Sciences)) and eluted. The elution pattern of size exclusion chromatography was measured with a wavelength of 280 nm and collected using the Unicorn program (GE Healthcare Life Sciences).

As shown in Figure 3b, the final purification of the encapsulin protein expressed in water-soluble high efficiency in E. coli was confirmed, and the concentration of the finally purified protein was BSA (Amresco, Solon, OH, USA) using BSA (Amresco, Solon, OH, USA). quantified.

<Example 1-4>

In order to confirm the overall diameter distribution of the fusion protein (VLP) including the purified encapsulin protein, dynamic light scattering (DLS) was used. Analysis of the complex was measured by putting 1 mL of a sample in a cuvette at a temperature of 16°C using DLS (Particular Systems, Zetasizer Nano Family). Dynamic light scattering analysis of cells was confirmed using the total intensity and mass of each area in the image. As shown in FIGS. 3c and 3d, it was confirmed that the diameter in the Hydrodynamic Radius of DLS appeared to be similar to that of 30-40 nm in which wild-type encapsulin (Encapsulin) appeared.

<Example 1-5>

The appearance of the fusion protein (VLP) containing the purified encapsulin protein was observed with an electron microscope. The purified Encapsulin VLPs were first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, dried at room temperature for 10 minutes, and then under a transmission electron microscope (TEM, Transmission electron microscope (120 kV)). ; Talos L120C, FEI, Czech) was used. As a result of the above results, as shown in Figure 3e, it was confirmed that the purified encapsulin (Encapsulin) protein to form a VLP. The diameter of the confirmed VLP was confirmed between 30 and 40 nm, which was confirmed to be similar to the diameter of the wild-type encapsulin (Encapsulin).

[Example 2]

Preparation of encapsulin rotavirus antigen complex with the addition of N-terminal expression level and water solubility enhancing partner for high efficiency and water solubility enhancement

<Example 2-1>

A rotavirus antigen, VP8* (VP8 core region), was inserted to create an encapsulin-based virus-like three-dimensional structure composed of an encapsulin protein fusion protein with a target protein attached to the C-terminus. For the inserted rotavirus, the VP8 core region (Human rotavirus A strain Wa G1P[8], (NCBI access number: FJ423116) gene derived from Rota virus G1P[8] was used (SEQ ID NO: 2). VP8, an antigen of rotavirus) * is composed of 159 amino acids, and the molecular weight of the protein corresponds to about 18.3 kDa Based on the protein sequence, a synthesized gene sequence was prepared after codon optimization in E. coli (SEQ ID NO: 2). Encapsulin gene synthesis request)

In Figure 4a, the VP8* antigen of the rotavirus was attached to the C-terminus of the encapsulin, and then the expression level and water solubility were confirmed. In addition, the effect on the expression of the fusion protein was confirmed by applying RID, a fusion partner to enhance the expression level and water solubility, to the N-terminus of the encapsulin protein and the VP8* antigen fusion protein to enhance high efficiency and water solubility. For the above purpose, MSAVKAA (SEQ ID NO: 5) and RID peptide (SEQ ID NO: 6) and mutant RID peptide (SEQ ID NO: 7) having the effect of increasing expression level and water solubility for the above purpose at the N-terminus of the recombinant protein was prepared. For the mutant RID used in the expression of this experiment to increase water solubility, amino acids of K32A, K27A, K31A, K23A/K27A, K23A/K31A of wild-type RID were substituted. In addition, (GaSb)n (a>1, b>1, n>1), which is a linker of the optimized sequence by applying ENLYFQG/S, which is the sequence of the TEV cleavage site, was combined with the TEV cleavage sequence and the fusion protein sequence. put in between A schematic diagram of the fusion protein expression vector constructed in this Example is shown in FIG. 4B.

The gene of the above sequence was prepared based on the pET-9a-Encapsulin vector, the recombinant complex protein expression vector prepared in Example 1, using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO: 4) expression vector. T7 RNA polymerase is expressed by IPTG, through which the Lac operon present on the DE3 genome and the existing encapsulin inserted into the MCS (Multiple Cloning Site) of the pET vector in which the T7 promoter operates. A RID sequence for increasing water solubility and expression rate was added to the N-terminus of the complex protein vector sequence, and a linking site sequence was added to enhance TEV cleavage efficiency. The pET9a vector was cut out using Nde1, KpnI, and BamHI cleavage enzymes, subcloned, and reinserted. Among the cleavage enzyme sites present inside the MCS, the RID and linking site (Linker1) of this study, the cleavage enzyme site, and the linking site (Linker2) to increase the cleavage efficiency of the TEV enzyme were inserted between the NdeI and KpnI sequences, and KpnI and BamHI DNA sequences designed to have a protein in the form of a fusion of encapsulin and rotavirus antigen protein were inserted between the cleavage enzymes, and each gene was inserted into the pET vector and the inserted gene through T4 DNA ligase. Sequences were ligated, and it was confirmed that the codon-optimized nucleotide sequence was inserted through DNA sequencing in the bacterial clones generated due to their DNA ligation reaction.

The sequence of the recombinant complex expression vector prepared in Example 2-1 is as follows.

ENC-Linker-VP8* [(SEQ ID NO: 1)-(SEQ ID NO: 10)-(SEQ ID NO: 2)]

: MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF-GGGSGGGSHHHHHHGGGS-LDGPYQPTTFTPPTDYWILINSNTNGVVYESTNNSDFWTAVIAVEPHVNPVDRQYNVFGENKQFNVRNDSDKWKFLEMFRGSSQNDFYNRRTLTSDTRLVGILKYGGRIWTFHGETPRATTDSSNTANLNGISITIHSEFYIIPRSQESKCNEYINNGL

The prepared complex was named "ENC-VP8*".

MSAVKAA-RID-Linker1-TEV-Linker2-ENC-Linker3-VP8* [(SEQ ID NO:5)-(SEQ ID NO:7)-(SEQ ID NO:8)-TEV-(SEQ ID NO:9)-(SEQ ID NO:1)-( SEQ ID NO:10)-(SEQ ID NO:2)]

: MSAVKAA-AAVQAAEVKVDGSEPKLSANELAARLAAEAAVAEAEAAQAELSEKQLSQATAAATNHTTDNGVGPEEESV-GGGSGGGS-ENLYFQ-GSGSGS-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF-GGGSGGGSHHHHHHGGGS-LDGPYQPTTFTPPTDYWILINSNTNGVVYESTNNSDFWTAVIAVEPHVNPVDRQYNVFGENKQFNVRNDSDKWKFLEMFRGSSQNDFYNRRTLTSDTRLVGILKYGGRIWTFHGETPRATTDSSNTANLNGISITIHSEFYIIPRSQESKCNEYINNGL

The prepared complex was named "RID-ENC-VP8*".

<Example 2-2>

The expression vector prepared in Example 2-1 was transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed E. coli were cultured in 3 ml of LB medium containing 50 μg/ml kanamycin at 37° C. at 250 rpm for 5-7 hours, then 1 ml was transferred to 10 ml, diluted 10 times, and further cultured. After about 2-4 hours, when O.D600 0.8~0.9 was reached, 0.4mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added and 17-19 hours at 16°C ( up to 21 hours). As the culture medium, only the precipitated E. coli was obtained through centrifugation and stored frozen at -80°C. 0.3 ml of lysis buffer solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 10 mM imidazole) was added to the E. coli harvest corresponding to 3 ml of culture medium and sonicated After grinding, the whole lysate (T, Total lysate) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) through centrifugation, and then analyzed by SDS-PAGE. The results are as shown in FIG. 4C. It was confirmed that the recombinant encapsulin protein prepared in Example 2-1 was highly soluble in water-soluble expression in E. coli. It was confirmed that Enc-VP8* and RID-ENC-VP8* were also expressed at appropriate positions corresponding to their respective sizes. However, the protein in which the rotavirus antigen is fused to encapsulin has a size of 50.3 kDa and when expressed by fusion of a peptide or protein of a certain size or more to the C-terminus, it can be confirmed that the solubility of the fusion protein is significantly reduced. there was. Therefore, in order to prepare a VLP polymer finally exposed to a foreign antigen surface through fusion expression of a foreign antigen protein to the C-terminus of an encapsulin derived from Thermotoga Maritama, first, high expression and water solubility of the fusion protein A water-soluble expression marker that significantly increases the expression tag (Soluble Expression Tag) is essential. In the present invention, RID (RNA Interaction Domain), which significantly increases the high expression level and water solubility, is fused to the N-terminus of encapsulin as a water-soluble expression marker, and a large molecular weight (about 18 kDa or more) is fused to the C-terminus. A high-efficiency water-soluble expression of a fusion protein fused with a foreign antigen of As a result, as shown in FIG. 4c , when expressing a single protein encapsulin, the protein has high water solubility, but when the antigen protein is fused to the C-terminus of the encapsulin, It was confirmed that a protein with high water solubility was obtained only when a water-soluble marker expression factor was added to the N-terminus and expressed. In addition, when a ligation site sequence of an appropriate length and sequence was added after the enzyme cleavage sequence, the TEV enzyme cleavage efficiency was as high as 90% or more, making it possible to purify the final protein in high yield in the subsequent purification process.

<Example 2-3>

Cells obtained by culturing and inducing expression of 500 ml of E. coli using the same culture method as in Example 2-2, in which high-efficiency water-soluble expression was confirmed, were mixed with 75 ml of lysis buffer solution [50 mM Tris-HCl A buffer [50 mM Tris-HCl]. (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 10 mM imidazole]. The cell resuspension was disrupted using an ultrasonic cell disrupter (sonication: pulse 3s/17s, 35% amplitude, 1min 30s, 4times total). The lysed cells were centrifuged at 13500 rpm at 4° C. for 10 minutes, and the supernatant was collected and Ni+-affinity chromatography was performed using AKTA (GE healthcare). First, the cation-exchange resin column HisTrap HP (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK, 5mL size) column was heated with buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.2 % Tween-20 and 10 mM imidazole), and then the aqueous solution containing the water-soluble protein was loaded onto the equilibrated column at a flow rate of 1 ml/min. Purification of RID-Encapsulin-VP8* protein was performed using buffer A and buffer B with 1M imidazole (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.2% Tween-20 and 1M imidazole). Imidazole was carried out in the form of increasing injection in a linear concentration gradient in the concentration range of 10mM to 1M using this, and through this, fractions of 2ml each were obtained.

After confirming the separation pattern of the target (target) protein through SDS-PAGE for the eluate, the eluate of the fraction was treated with Dialysis Buffer ([50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 0.2% Tween-20] ) was dialyzed at 9°C for 12-16 hours. At this time, after dialyzing first for 4 hours, the fusion protein in which the N-terminal fusion partner RID and the encapsulin protein and the rotavirus antigen are fused using TEV cleavage enzyme for the remaining 8-12 hours cut.

Biochemical analysis was performed to determine whether ENC-VP8* protein after IMAC and Dialysis forms VLP, and for subsequent purification, Ion Exchange Chromatography (Ion Exchange Chromatography) was performed using the AKTA FPLC system (GE healthcare). ) was performed. Encapsulin proteins obtained by the dialysis were purified by ion exchange resin chromatography using an FPLC system (AKTA, GE healthcare). Column (HiTrap Q FF, 1 mL size (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK)) is buffer A (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 5% glycerol, 0.2% Tween-20) was equilibrated with and loaded at a flow rate of 1 ml/min. ENC-VP8* protein was eluted by injection of NaCl having a linear concentration gradient of 10 mM to 1 M, and buffer solution A and buffer solution B (50 mM Tris-HCl (pH) 7.5), 1M NaCl, 5% glycerol, 0.2% Tween-20) was purified through a linear concentration gradient, and fractions of 1 ml each were obtained.Through this, other proteins derived from E. coli were purified fusion protein. (Fig. 4d) The purified ENC-VP8* protein was stored at 4 ° C with 20% glycerol, and the final concentration of the purified protein was BSA (Amresco, Solon, OH, USA). ) was used for quantification.

<Example 2-4>

의 온도 조건으로 샘플 1mL을 Cuvette에 넣어 측정하였다. 세포의 동적광산란 분석은 이미지에서 각 영역별 total intensity 및 Mass 등을 이용하여 확인하였다. 도 5a와 같이 DLS의 Hydrodynamic Radius에서의 직경 30~40nm의 Particle 크기의 분포를 갖는 것을 확인하였다.In order to confirm the overall diameter distribution of the fusion protein (VLP) composed of the ENC-VP8* fusion protein of Example 2-3, dynamic light scattering (DLS) was used. Analysis of the complex was performed using DLS (Particular Systems, Zetasizer Nano Family) 16

1mL of the sample was put into the cuvette under the temperature condition of Dynamic light scattering analysis of cells was confirmed using the total intensity and mass of each area in the image. As shown in Fig. 5a, it was confirmed that the particle size distribution of 30-40 nm in diameter in the Hydrodynamic Radius of DLS was obtained.

<Example 2-5>

The appearance of the virus-like stereotactic (VLP) formed by the purified encapsulin protein was observed with an electron microscope. The purified Encapsulin VLPs were first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, dried at room temperature for 10 minutes, and then under a transmission electron microscope (TEM, Transmission electron microscope (120 kV)). ; Talos L120C, FEI, Czech) was used. As shown in Figure 5b, it was confirmed that the purified ENC-VP8* protein forms VLP. The diameter of the confirmed VLP appears to be between 30 and 40 nm, which is the diameter of the existing encapsulin protein, and it was confirmed that a spherical VLP was also formed in the form of a fusion protein in which an antigen protein was fused to the C-terminus.

<Example 2-6>

In order to measure the antigen-specific antibody inducibility, the finally purified ENC-VP8* fusion VLP protein was quantified using BSA (Amresco, Solon, OH, USA) and then added to an appropriate concentration with a buffer containing 20% glycerol. diluted. Antigen and adjuvant (Alhydrogel®, Invitrogen Cat# vac-alu-250)) were strongly mixed for more than 5 minutes using a syringe in a 1:1 ratio, and then 100ul in a 4-week-old BALB/C mouse group (6 mice/group). was inoculated via intramuscular injection. The vaccine was inoculated and immunized 3 times with 5ug or 10ug of ENC-VP8* VLP, respectively, at 2-week intervals, and serum was obtained through orbital blood sampling the day before vaccination. In order to measure the target protein-specific antibody in the serum obtained from each animal test group, an enzyme-linked immunoprecipitation assay (ELISA (enzyme-linked immunosorbent assay)) was performed. 100ul of the rotavirus VP8 antigen protein (P2-VP8) used as a control was placed in a 96-well plate (High Binding Capacity (HBC) NeutrAvidin plates (Thermo Scientific™)) at a concentration of 0.5ug/ml per well, 18 hours at 4 degrees After coating, 200ul of 3% BSA solution was added, and blocking was performed at 37°C for 2 hours. After washing the plate using PBST, the immune serum was diluted (1:1000 dilution ratio), 100ul per well, and incubated at 37°C for 1 hour and 30 minutes. After washing the plate using PBST, an antibody against goat-derived mouse IgG (southern Biotech 1030-05, Goat-anti mouse IgG HRP) was treated at 37°C for 1 hour to detect the antibody in the serum bound to P2-VP8*. After color development at room temperature for 10 minutes using TMB (sigma T0440) solution, the color reaction was stopped with 0.5 M sulfuric acid solution and absorbance was measured at 450 nm wavelength. As shown in Figure 6b, when the VP8-specific antibody titer of ENC-VP8* present in the first, second and third blood samples, respectively, was measured, high levels of VP8 in the 10ug Encapsulin_VP8_High as well as in the 5ug Encapsulin_VP8_Low group were measured. It was confirmed that a specific antibody was formed. In addition, in the purified peptide protein of the VP8-specific antibody titer control group, the antibody was confirmed from the tertiary 2nd Boost sera pool, while the formation of VP8-specific antibody was confirmed from the Boost sera pool, the secondary blood sampling sample, in both Encapsulin_VP8_High and Encapsulin_VP8_Low. did.

In addition, HBGA binding assay was performed to confirm whether ENC-VP8 binds to HBGA. As shown in 6c, in a 96-well plate (High Binding Capacity (HBC) NeutrAvidin plates (Thermo Scientific™)) coated with streptavidin, 100ul of HBGA with biotin at a concentration of 10ug/ml was put into each well and kept at room temperature 5 time was reacted. After washing the plate using PBST, ENC-VP8 protein was diluted in PBS and put into each well by 100ul, and reacted at 4°C for 18 hours. After washing the plate using PBST, mouse serum inoculated with ENC-VP8 was diluted at a ratio of 1:1000, 100ul per well, and reacted at room temperature for 1 hour. After washing with PBST, 5 antibodies against goat-derived mouse IgG (Goat-anti mouse IgG HRP) (H (type 2)-PAA-biotin (01-034), Lea-PAA-biotin (01-035), Led (H type1)-PAA-biotin (01-037), Blood type A (tri)-PAA-biotin (01-032), Blood type B(tri)-PAA-biotin (01-033); (Glycotech)) was treated at room temperature for 1 hour. After color development at room temperature using TMB solution for 10 minutes, the color development reaction was stopped with 0.5 M sulfuric acid solution, and absorbance was measured at 450 nm wavelength. As shown in Figure 6d, binding between HBGA and VP8 antigen was confirmed using primary sera obtained from the group injected with ENC-VP8*. As a result, the ENC-VP8* VLP fusion protein was found to bind to Led (H type 1) carbohydrate. Confirmed.

<Example 2-7>

In order to measure the degree to which immune serum inhibits the binding between the ENC-VP8* antigen and the receptor, HBGA blocking assay was performed (Fig. 6e). In a 96-well plate coated with streptavidin, HBGA with biotin was added to 100ul per well at a concentration of 10ug/ml and reacted at room temperature for 5 hours. ENC-VP8* was diluted to a concentration of 1 ug/ml and then reacted with immune serum at a ratio of 1:1 at 37°C for 3 hours. Then, 100ul of serum and ENC-VP8* reaction solution were added per well to the plate reacted with HBGA washed with PBST, and reacted at 4°C for 18 hours. After washing the plate using PBST, mouse serum inoculated with ENC-VP8* was diluted at a ratio of 1:1000, 100ul per well, and reacted at room temperature for 1 hour. After washing with PBST, an antibody against goat-derived mouse IgG (Goat-anti mouse IgG HRP) was treated at room temperature for 1 hour. After that, the color was developed at room temperature using TMB solution for 10 minutes, the color reaction was stopped with 0.5 M sulfuric acid solution, and the absorbance was measured at a wavelength of 450 nm. As shown in Figure 6f, after coating Led (H type 1) on a plate with Led (H type 1) that confirmed the binding of ENC-VP8 VLP antigen protein in HBGA Binding Assay, Encapsulin_VP8 VLP was diluted with various concentrations of immune sera ( primary, boost, and 2nd boost) and the binding to Led was measured by ELISA. As a result, in the case of the ENC-VP8 antigen fusion VLP protein of this example, in both EncVP8_High and Low groups, the immune serum from the primary sera was confirmed, and as the inoculation was repeated until the third inoculation, the inhibitory ability of immune serum was significantly increased compared to the control group. On the other hand, in the control group, P2 VP8 group, immune sera inhibited antigen-receptor binding from the 2nd immune sera after the second vaccination, and it was confirmed that the protective effect was lower than that of the Enc_VP8 group even after the third vaccination.

Through the results of Example 2, when the VLP in which Encapsulin and the target protein according to the present invention were fused, a much higher level of the target protein-specific antibody was induced earlier than when only the target protein was administered. confirmed that the performance of inhibiting the binding between the target protein and its receptor is excellent.

[Example 3]

Preparation of encapsulin human papilloma antigen complex with N-terminal peptide added for high efficiency and water solubility enhancement

<Example 3-1>

In order to examine whether it is possible to form a VLP composed of a virus-like stereotype based on the fusion protein encapsulin when a protein smaller than the rotavirus VP8* antigen of about 18.3 kDa in size consisting of 159 amino acids is inserted at the C-terminus. (HPV) antigen was used. E7 LP, a human papillomavirus antigen, was inserted as an encapsulin protein, and the E7 core region (E7 protein, partial [Human papillomavirus type 16] Sequence ID: ABL96584.1) gene derived from human papillomavirus HPV 16 was used ( SEQ ID NO: 3). The antigen of human papillomavirus, E7 LP (Long peptide), consists of 35 amino acids and has a size of about 4 kDa. Based on the protein sequence, a gene sequence synthesized after codon optimization in E. coli was prepared (SEQ ID NO: 3).

(Request for Encapsulin gene synthesis to Bionics Co., Ltd.):

Human papillomavirus E7 LP was inserted at the C-terminus of the encapsulin protein. In addition, as shown in FIG. 8a , the effect on the expression of the fusion protein was confirmed by applying RID, a fusion partner to enhance the expression level and water solubility, to the N-terminus of the encapsulin protein to enhance high efficiency and water solubility. Accordingly, MSAVKAA (SEQ ID NO: 5) and RID peptide (SEQ ID NO: 6) and a mutant RID peptide (SEQ ID NO: 7) having at least 50% similarity at the N-terminus were prepared for increased expression and water solubility. . For the mutant RID used in the expression of this experiment to increase water solubility, amino acids of K32A, K27A, K31A, K23A/K27A, K23A/K31A of wild-type RID were substituted. In addition, (GaSb)n (a>1, b>1, n>1), which is a linker of the optimized sequence by applying ENLYFQG/S, which is the sequence of the TEV cleavage site, was combined with the TEV cleavage sequence and the fusion protein sequence. put in between The fusion protein expression vector constructed in this Example is shown in FIG. 7B.

The gene of the above sequence was pET9a-Encapsulin-VP8* vector and pET9a-MSAVKAA-RID, the recombinant complex protein expression vector prepared in Example 2 using the pET-9a (Novagene, 69431-3CN) (SEQ ID NO: 4) expression vector. -Linker-Enc-Linker-VP8* Vector was created based on the vector, and the rotavirus antigen protein at the C-terminus of Enc was replaced with a human papillomavirus antigen using NcoI and BamHI cleavage enzymes and inserted.

The sequence of the recombinant complex expression vector prepared in Example 3-1 is as follows.

ENC-Linker-E7 LP[(SEQ ID NO: 1)-(SEQ ID NO: 10)-(SEQ ID NO: 3)]

MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF-GGGSGGGSHHHHHHGGGS- GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR

[Encapsulin-Linker-HPV E7] [(SEQ ID NO: 1)- (SEQ ID NO: 10)-(SEQ ID NO: 3)]

The prepared complex was named "Enc-E7 LP".

MSAVKAA-RID-Linker1-TEV-Linker2-ENC-Linker-E7 LP

[(SEQ ID NO: 5)-(SEQ ID NO: 6)-(SEQ ID NO: 8)-TEV-(SEQ ID NO: 9)-(SEQ ID NO: 1)-(SEQ ID NO: 10)-(SEQ ID NO: 3)]

:MSAVKAA-AAVQAAEVKVDGSEPKLSKNELKRRLKAEKKVAEKEAKQKELSEKQLSQATAAATNHTTDNGVGPEEESV

-ENLYFQ-GSGSGS-MEFLKRSFAPLTEKQWQEIDNRAREIFKTQLYGRKFVDVEGPYGWEYAAHPLGEVEVLSDENEVVKWGLRKSLPLIELRATFTLDLWELDNLERGKPNVDLSSLEETVRKVAEFEDEVIFRGCEKSGVKGLLSFEERKIECGSTPKDLLEAIVRALSIFSKDGIEGPYTLVINTDRWINFLKEEAGHYPLEKRVEECLRGGKIITTPRIEDALVVSERGGDFKLILGQDLSIGYEDREKDAVRLFITETFTFQVVNPEALILLKF-GGGSGGGSHHHHHHGGGS- GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR

The prepared complex was named "RID-ENC-E7 LP". (LP stands for Long Peptide.)

<Example 3-2>

The expression vector prepared in Example 3-1 was transformed into HMS174 competent cells (Novagen (RecA mutation in a K-12 strain, Cat: 69453-3)). All transformed E. coli were incubated in 3 ml of LB medium containing 50 μg/ml kanamycin at 37 ° C. at 250 rpm for 5-7 hours, then 1 ml was transferred to 10 ml, diluted 10 times, and then further cultured. After about 2-4 hours, when O.D600 0.8~0.9 was reached, 0.4mM IPTG (Biosesang, Cat#I1006, Lot#LB0C0340, cas#367-93-1) was added and 17-19 hours at 16°C ( up to 21 hours). The culture medium obtained only the precipitated E. coli through centrifugation and stored frozen at -80 ° C. 0.3 ml of lysis buffer solution (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 10 mM imidazole) was added to the E. coli harvest corresponding to 3 ml of culture medium and sonicated After grinding, the total lysate (T, Total lysate) was separated into a precipitate (P, Precipitant) and a supernatant (S, Soluble lysate) through centrifugation, and then analyzed by SDS-PAGE. As a result, as shown in FIGS. 7c and 7d , it was confirmed that ENC-E7 LP and RID-ENC-E7 LP corresponding to each size were expressed and confirmed that 36.1 kDa and 45.7 kDa proteins were expressed at appropriate positions. However, it was confirmed that the solubility of the fusion protein was remarkably reduced even when the human papillomavirus of 35 amino acids and 4kDa smaller than the 18kDa rotavirus was fused to the C-terminus of the encapsulin. It was confirmed that RID, a high-efficiency and water-soluble enhancing fusion partner, is required to be expressed in E. coli as an encapsulin complex, ranging from peptides of sequence or small-sized to large-sized proteins. Therefore, as in Example 2, in order to prepare a VLP polymer finally exposed to the surface of the foreign antigen through fusion expression of the foreign antigen protein to the C-terminus of the encapsulin, the water-soluble expression marker of the present invention It was reconfirmed that is essential, and the need in the E. coli expression system of RID (RNA Interaction Domain) and water-soluble expression markers, which significantly increases the high expression level and water solubility in the present invention, was demonstrated.

<Example 3-3>

The RID-ENC-E7 LP complex protein prepared using the same culture method as in Example 3-2, whose water solubility was confirmed, was purified through nickel (Ni) affinity chromatography. Cells obtained by culturing and inducing expression of 500 ml of E. coli using the same culture method were treated with 75 ml of lysis buffer [A buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.1% Triton X-100 and 10 mM imidazole]. The cell resuspension was disrupted using an ultrasonic cell disrupter (sonication: pulse 3s/17s, 35% amplitude, 1min 30s, 4times total). The lysed cells were centrifuged at 13500 rpm at 4° C. for 10 minutes, and the supernatant was collected and Ni+-affinity chromatography was performed using AKTA (GE healthcare). First, the cation-exchange resin column HisTrap HP (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK, 5mL size) column was heated with buffer A (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.2 % Tween-20 and 10 mM imidazole), and then the aqueous solution containing the water-soluble protein was loaded onto the equilibrated column at a flow rate of 1 ml/min. Purification of RID-Encapsulin-VP8* protein was performed using buffer A and buffer B with 1M imidazole (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, 0.2% Tween-20 and 1M imidazole). Imidazole was carried out in the form of increasing injection in a linear concentration gradient in the concentration range of 10mM to 1M using this, and through this, fractions of 2ml each were obtained.

As shown in FIGS. 8a and 8b, after confirming the separation pattern of the target (target) protein through SDS-PAGE for the eluate, the eluate of the fraction was treated with a Dialysis buffer ([50 mM Tris-HCl (pH 8.5), 10 mM NaCl, 0.2% Tween-20]) at 9° C. for 12-16 hours. At this time, after dialyzing first for 4 hours, the fusion protein in which the N-terminal fusion partner RID and the encapsulin protein and the human papillomavirus antigen are fused is cut using a TEV cleaving enzyme for the remaining 8-12 hours. did.

에서 보관하였으며, 최종적으로 정제된 단백질의 농도는 BSA(Amresco, Solon, OH, USA)를 이용하여 정량 하였다. 도 9a에 보여지는 바와 같이, 본 발명의 RID 및 수용성을 위한 추가 표지 인자가 단백질 발현부터 TEV 효소 처리 및 이온 교환 수지 크로마토그래피에 이르기까지의 정제 과정에서의 융합 단백질의 정제 양상 및 순도를 확인하였으며, 인유두종 바이러스의 항원의 경우에도 단백질을 안정적으로 획득하는 데 필수임을 확인하였다.Biochemical analysis was performed to determine whether the ENC-E7 LP protein after IMAC and Dialysis forms VLP, and for subsequent purification, Ion Exchange Chromatography (Ion Exchange Chromatography) was performed using the AKTA FPLC system (GE healthcare). ) was performed. Encapsulin proteins obtained by the above dialysis were purified by ion exchange resin chromatography using an FPLC system (AKTA, GE healthcare). Column (HiTrap Q FF, 1 mL size (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK)) is buffer A (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 5% glycerol, 0.2% Tween-20) was equilibrated with and loaded at a flow rate of 1 ml/min Encapsulin-E7 LP protein was eluted by injecting NaCl having a linear concentration gradient of 10 mM to 1 M, and buffer solution A and buffer solution B (50 mM Tris-HCl (pH) 7.5), 1M NaCl, 5% glycerol, 0.2% Tween-20) was purified through a linear concentration gradient, and fractions of 1 ml each were obtained.Through this, other proteins derived from E. coli were purified fusion protein. (Fig. 8d) Purified ENC-E7 LP protein was 4 in a state containing 20% glycerol

, and finally the purified protein concentration was quantified using BSA (Amresco, Solon, OH, USA). As shown in Figure 9a, additional markers for RID and water solubility of the present invention confirmed the purification aspect and purity of the fusion protein in the purification process from protein expression to TEV enzyme treatment and ion exchange resin chromatography. , it was confirmed that even in the case of an antigen of human papillomavirus, it is essential for stably acquiring the protein.

<Example 3-4>

In order to confirm the overall diameter of the purified virus-like particles (VLPs) of the ENC-E7 LP complex of Example 3-3, dynamic light scattering (DLS) was used. Analysis of the complex was measured by putting 1 mL of a sample in a cuvette at a temperature of 16°C using DLS (Particular Systems, Zetasizer Nano Family). Dynamic light scattering analysis of cells was confirmed using the total intensity and mass of each area in the image. As shown in FIG. 9b, it was confirmed that the diameter in the Hydrodynamic Radius of DLS had a distribution of a size similar to that of 30-40 nm that wild-type encapsulin was seen.

<Example 3-5>

The appearance of the virus-like stereotactic (VLP) formed by the purified encapsulin protein was observed with an electron microscope. The purified Encapsulin VLPs were first placed on a copper grid for 1 minute, then stained with 2% uranyl acetate for 1 minute, dried at room temperature for 10 minutes, and then under a transmission electron microscope (TEM, Transmission electron microscope (120 kV)). ; Talos L120C, FEI, Czech) was used. As shown in Figure 9c, it was confirmed that the purified encapsulin (Encapsulin) protein to form a VLP.

As a result of the above obtained results, as shown in Figure 9c, it was confirmed that the purified encapsulin (Encapsulin) protein to form a VLP. The diameter of the confirmed VLP appears to be between 30 and 40 nm, which is the diameter of the existing encapsulin protein, and it was confirmed that a spherical VLP was also formed in the form of a fusion protein in which an antigen protein was fused to the C-terminus. In the results of Example 3, as in the results of Example 2, both high-efficiency protein production and VLP formation in E. coli of Encapsulin using the present invention were confirmed, which is RID, a high-efficiency and water-soluble enhancement fusion partner of this experiment. indicates that it is essential to form a fusion protein-based platform in which a protein such as an antigen is inserted at the C-terminus of the encapsulin.

Although embodiments of the present invention have been described above, those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without changing the technical spirit or essential features thereof. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> InThera Inc. <120> RECOMBINANT EXPRESSION VECTOR FOR PRODUCTION OF ENCAPSULIN-BASED VACCINE AND METHOD FOR MANUFACTURING THE SAME <130> 1067575 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 265 <212> PRT <213> Artificial Sequence <220> <223> ENCAPSULIN <400> 1 Met Glu Phe Leu Lys Arg Ser Phe Ala Pro Leu Thr Glu Lys Gln Trp 1 5 10 15 Gln Glu Ile Asp Asn Arg Ala Arg Glu Ile Phe Lys Thr Gln Leu Tyr 20 25 30 Gly Arg Lys Phe Val Asp Val Glu Gly Pro Tyr Gly Trp Glu Tyr Ala 35 40 45 Ala His Pro Leu Gly Glu Val Glu Val Leu Ser Asp Glu Asn Glu Val 50 55 60 Val Lys Trp Gly Leu Arg Lys Ser Leu Pro Leu Ile Glu Leu Arg Ala 65 70 75 80 Thr Phe Thr Leu Asp Leu Trp Glu Leu Asp Asn Leu Glu Arg Gly Lys 85 90 95 Pro Asn Val Asp Leu Ser Ser Leu Glu Thr Val Arg Lys Val Ala 100 105 110 Glu Phe Glu Asp Glu Val Ile Phe Arg Gly Cys Glu Lys Ser Gly Val 115 120 125 Lys Gly Leu Leu Ser Phe Glu Glu Arg Lys Ile Glu Cys Gly Ser Thr 130 135 140 Pro Lys Asp Leu Leu Glu Ala Ile Val Arg Ala Leu Ser Ile Phe Ser 145 150 155 160 Lys Asp Gly Ile Glu Gly Pro Tyr Thr Leu Val Ile Asn Thr Asp Arg 165 170 175 Trp Ile Asn Phe Leu Lys Glu Glu Ala Gly His Tyr Pro Leu Glu Lys 180 185 190 Arg Val Glu Glu Cys Leu Arg Gly Gly Lys Ile Ile Thr Thr Pro Arg 195 200 205 Ile Glu Asp Ala Leu Val Val Ser Glu Arg Gly Gly Asp Phe Lys Leu 210 215 220 Ile Leu Gly Gln Asp Leu Ser Ile Gly Tyr Glu Asp Arg Glu Lys Asp 225 230 235 240 Ala Val Arg Leu Phe Ile Thr Glu Thr Phe Thr Phe Gln Val Val Asn 245 250 255 Pro Glu Ala Leu Ile Leu Leu Lys Phe 260 265 <210> 2 <211> 159 <212> PRT <213> Artificial Sequence <220> <223> Rotavirus G1P[8] VP8* <400> 2 Leu Asp Gly Pro Tyr Gln Pro Thr Thr Phe Thr Pro Pro Thr Asp Tyr 1 5 10 15 Trp Ile Leu Ile Asn Ser Asn Thr Asn Gly Val Val Tyr Glu Ser Thr 20 25 30 Asn Asn Ser Asp Phe Trp Thr Ala Val Ile Ala Val Glu Pro His Val 35 40 45 Asn Pro Val Asp Arg Gln Tyr Asn Val Phe Gly Glu Asn Lys Gln Phe 50 55 60 Asn Val Arg Asn Asp Ser Asp Lys Trp Lys Phe Leu Glu Met Phe Arg 65 70 75 80 Gly Ser Ser Gln Asn Asp Phe Tyr Asn Arg Arg Thr Leu Thr Ser Asp 85 90 95 Thr Arg Leu Val Gly Ile Leu Lys Tyr Gly Gly Arg Ile Trp Thr Phe 100 105 110 His Gly Glu Thr Pro Arg Ala Thr Thr Asp Ser Ser Asn Thr Ala Asn 115 120 125 Leu Asn Gly Ile Ser Ile Thr Ile His Ser Glu Phe Tyr Ile Ile Pro 130 135 140 Arg Ser Gln Glu Ser Lys Cys Asn Glu Tyr Ile Asn Asn Gly Leu 145 150 155 <210> 3 <211> 35 <212> PRT <213> Artificial Sequence <220> <223> HPV E7 <400> 3 Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys 1 5 10 15 Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val 20 25 30 Asp Ile Arg 35 <210> 4 <211> 4341 <212> DNA <213> Artificial Sequence <220> <223> Vector Map (pET-9a) <400> 4 ttctcatgtt tgacagctta tcatcgataa gctttaatgc ggtagtttat cacagttaaa 60 ttgctaacgc agtcaggcac cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg 120 caccgtcacc ctggatgctg taggcatagg cttggttatg ccggtactgc cgggcctctt 180 gcgggatatc gtccattccg acagcatcgc cagtcactat ggcgtgctgc tagcgctata 240 tgcgttgatg caatttctat gcgcacccgt tctcggagca ctgtccgacc gctttggccg 300 ccgcccagtc ctgctcgctt cgctacttgg agccactatc gactacgcga tcatggcgac 360 cacacccgtc ctgtggatat ccggatatag ttcctccttt cagcaaaaaa cccctcaaga 420 cccgtttaga ggccccaagg ggttatgcta gttattgctc agcggtggca gcagccaact 480 cagcttcctt tcgggctttg ttagcagccg gatccgcgac ccatttgctg tccaccagtc 540 atgctagcca tatgtatatc tccttcttaa agttaaacaa aattatttct agagggaaac 600 cgttgtggtc tccctatagt gagtcgtatt aatttcgcgg gatcgagatc tcgatcctct 660 acgccggacg catcgtggcc ggcatcaccg gcgccacagg tgcggttgct ggcgcctata 720 tcgccgacat caccgatggg gaagatcggg ctcgccactt cgggctcatg agcgcttgtt 780 tcggcgtggg tatggtggca ggccccgtgg ccgggggact gttgggcgcc atctccttgc 840 atgcaccatt ccttgcggcg gcggtgctca acggcctcaa cctactactg ggctgcttcc 900 taatgcagga gtcgcataag ggagagcgtc gaccgatgcc cttgagagcc ttcaacccag 960 tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc cgcacttatg actgtcttct 1020 ttatcatgca actcgtagga caggtgccgg cagcgctctg ggtcatttt ggcgaggacc 1080 gctttcgctg gagcgcgacg atgatcggcc tgtcgcttgc ggtattcgga atcttgcacg 1140 ccctcgctca agccttcgtc actggtcccg ccaccaaacg tttcggcgag aagcaggcca 1200 ttatcgccgg catggcggcc gacgcgctgg gctacgtctt gctggcgttc gcgacgcgag 1260 gctggatggc cttccccatt atgattcttc tcgcttccgg cggcatcggg atgcccgcgt 1320 tgcaggccat gctgtccagg caggtagatg acgaccatca gggacagctt caaggatcgc 1380 tcgcggctct taccagccta acttcgatca ctggaccgct gatcgtcacg gcgatttatg 1440 ccgcctcggc gagcacatgg aacgggttgg catggattgt aggcgccgcc ctataccttg 1500 tctgcctccc cgcgttgcgt cgcggtgcat ggagccgggc cacctcgacc tgaatggaag 1560 ccggcggcac ctcgctaacg gattcaccac tccaagaatt ggagccaatc aattcttgcg 1620 gagaactgtg aatgcgcaaa ccaacccttg gcagaacata tccatcgcgt ccgccatctc 1680 cagcagccgc acgcggcgca tctcgggcag cgttgggtcc tggccacggg tgcgcatgat 1740 cgtgctcctg tcgttgagga cccggctagg ctggcggggt tgccttactg gttagcagaa 1800 tgaatcaccg atacgcgagc gaacgtgaag cgactgctgc tgcaaaacgt ctgcgacctg 1860 agcaacaaca tgaatggtct tcggtttccg tgtttcgtaa agtctggaaa cgcggaagtc 1920 agcgccctgc accattatgt tccggatctg catcgcagga tgctgctggc taccctgtgg 1980 aacacctaca tctgtattata cgaagcgctg gcattgaccc tgagtgattt ttctctggtc 2040 ccgccgcatc cataccgcca gttgtttacc ctcacaacgt tccagtaacc gggcatgttc 2100 atcatcagta acccgtatcg tgagcatcct ctctcgtttc atcggtatca ttacccccat 2160 gaacagaaat cccccttaca cggaggcatc agtgaccaaa caggaaaaaa ccgcccttaa 2220 catggcccgc tttatcagaa gccagacatt aacgcttctg gagaaactca acgagctgga 2280 cgcggatgaa caggcagaca tctgtgaatc gcttcacgac cacgctgatg agctttaccg 2340 cagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga 2400 gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc 2460 agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt 2520 gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatatgc 2580 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt 2640 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 2700 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 2760 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 2820 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 2880 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 2940 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 3000 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 3060 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 3120 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 3180 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 3240 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 3300 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 3360 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 3420 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca 3480 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg 3540 gaaacgtctt gctcgaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat 3600 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag 3660 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca 3720 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat 3780 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca 3840 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg 3900 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta 3960 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt 4020 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taagcttttg 4080 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt 4140 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac 4200 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg 4260 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg 4320 ctcgatgagt ttttctaaga a 4341 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> MSAVKAA <400> 5 Met Ser Ala Val Lys Ala Ala 1 5 <210> 6 <211> 70 <212> PRT <213> Artificial Sequence <220> <223> ARSNTD <400> 6 Ala Ala Val Gln Ala Ala Glu Val Lys Val Asp Gly Ser Glu Pro Lys 1 5 10 15 Leu Ser Lys Asn Glu Leu Lys Arg Arg Leu Lys Ala Glu Lys Lys Val 20 25 30 Ala Glu Lys Glu Ala Lys Gln Lys Glu Leu Ser Glu Lys Gln Leu Ser 35 40 45 Gln Ala Thr Ala Ala Ala Thr Asn His Thr Thr Asp Asn Gly Val Gly 50 55 60 Pro Glu Glu Glu Ser Val 65 70 <210> 7 <211> 70 <212> PRT <213> Artificial Sequence <220> <223> MARSNTD(9) <400> 7 Ala Ala Val Gln Ala Ala Glu Val Lys Val Asp Gly Ser Glu Pro Lys 1 5 10 15 Leu Ser Ala Asn Glu Leu Ala Ala Arg Leu Ala Ala Glu Ala Ala Val 20 25 30 Ala Glu Ala Glu Ala Ala Gln Ala Glu Leu Ser Glu Lys Gln Leu Ser 35 40 45 Gln Ala Thr Ala Ala Ala Thr Asn His Thr Thr Asp Asn Gly Val Gly 50 55 60 Pro Glu Glu Glu Ser Val 65 70 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> LINKER1 <400> 8 ggcggtggct ctggcggtgg ctct 24 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> LINKER2 <400> 9 ggttctggtt ctggttct 18 <210> 10 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> LINKER <400> 10 ggcggtggct ctggcggtgg ctctcaccat caccatcacc atggcggtgg ctct 54

Claims (24)

delete delete delete delete delete delete target protein;
encapsulin protein; and
Contains a polynucleotide encoding an RNA interacting domain (RID) protein;
The target protein is a rotavirus antigen protein or a cervical cancer virus antigen protein, a recombinant expression vector for vaccine production. 8. The method of claim 7,
The encapsulin protein is a recombinant expression vector for vaccine production, characterized in that it comprises the amino acid sequence of SEQ ID NO: 1. 8. The method of claim 7,
The RID protein is a recombinant expression vector for vaccine production, characterized in that it is a fusion partner for increasing the expression level or water solubility of the vaccine. 8. The method of claim 7,
The RID protein is a recombinant expression vector for vaccine production, characterized in that it comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. 8. The method of claim 7,
The polynucleotide encodes a TEV protein for cleavage of the RID protein, a recombinant expression vector for vaccine production. delete delete A host cell transformed with the expression vector according to any one of claims 7 to 11. 15. The method of claim 14,
The host cell is a transformed host cell, characterized in that E. coli (E. coli). target protein; encapsulin protein; and an RNA interacting domain (RID) protein;
The target protein is a rotavirus antigen protein or a cervical cancer virus antigen protein, a fusion protein. 17. The method of claim 16,
The target protein is a fusion protein, characterized in that linked to the C-terminus of the encapsulin protein. a) the protein of interest; encapsulin protein; And RID protein; producing a recombinant expression vector for vaccine production comprising a polynucleotide encoding the;
b) introducing the expression vector into a host cell to produce a transformant; and
c) inducing expression of the recombinant fusion protein by culturing the transformant, and obtaining it;
The target protein is a rotavirus antigen protein or a cervical cancer virus antigen protein, a vaccine production method. 19. The method of claim 18,
The RID protein is a vaccine production method, characterized in that the fusion partner for increasing the expression level or water solubility of the vaccine. 19. The method of claim 18,
The RID protein is a vaccine production method, characterized in that it comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. 19. The method of claim 18,
The host cell is Escherichia coli ( E. coli ) Vaccine production method, characterized in that. 19. The method of claim 18,
The encapsulin protein is a vaccine production method, characterized in that it comprises the amino acid sequence of SEQ ID NO: 1.
delete delete

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