Preparation of flagellin vaccine adjuvant-based vaccine to induce production of antibody recognizing conformation of antigens, and application thereof

Abstract

The present invention provides a vaccine composition for use in neurodegenerative diseases and an infectious virus vaccine composition for inducing an antibody recognizing the conformation of antigens. The vaccine composition of the present invention induces the production of an antibody recognizing the conformation of antigens. The antibody recognizing the conformation of antigens has high specificity for an antigen, and thus can be useful for ameliorating, preventing or treating diseases.

Claims

1. A method for treatment of a neurodegenerative disease, the method comprising administering the composition containing a recombinant protein comprising: (a) a repeated domain (RD) of tau (T) protein; and (b) FlaB protein derived from Vibrio vulnificus, to a subject, wherein the FlaB protein has the amino acid sequence of SEQ ID NO: 2, and wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, tauopathy, dementia, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and memory loss.

2. The method of claim 1, wherein the RD has the amino acid sequence of SEQ ID NO: 3.

3. The method of claim 1, wherein the RD is encoded by the nucleotide sequence of SEQ ID NO: 5, which is a codon-optimized nucleotide sequence for expression in E. coli.

4. A method for providing information necessary for diagnosis of a neurodegenerative disease, the method comprising measuring a degree of production of an antibody produced by administering the composition containing a recombinant protein comprising: (a) a repeated domain (RD) of tau (T) protein; and (b) FlaB protein derived from Vibrio vulnificus, to a subject, wherein the FlaB protein has the amino acid sequence of SEQ ID NO: 2, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, tauopathy, dementia, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, and memory loss.

5. The method of claim 4, wherein the RD has the amino acid sequence of SEQ ID NO: 3.

6. The method of claim 4, wherein the RD is encoded by the nucleotide sequence of SEQ ID NO: 5, which is a codon-optimized nucleotide sequence for expression in E. coli.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 schematically shows a procedure of cloning a repeated domain (RD) of tau (τ) protein.

(2) FIG. 2 shows results confirming the cloning of RD of the tau protein. The left panel shows results confirming the expression of RD through SDS-PAGE and the right panel shows results confirming the expression of RD through western blotting. RD indicates a size of 13 kDa.

(3) FIG. 3 shows results confirming the cloning of FlaB-TauRD recombinant protein.

(4) FIG. 4 shows results confirming the production of a conformer structure recognizing antibody by administration of FlaB-TauRD recombinant protein. Experimental results show that an anti-serum obtained by the immunization of FlaB and Tau-Ag, which is obtained from the expression of a portion of Tau protein, induced the production of a “structure recognizing antibody” responding to a paired helical filament (PHF), which is a Tau pathologic conformer.

(5) FIG. 5 shows results confirming stimulation activity of FlaB, TauRD, and FlaB-TauRD to tall-like receptor 5 (TLR5).

(6) FIG. 6 shows an immunization schedule of FlaB-TauRD recombinant protein.

(7) FIGS. 7a and 7b show IgG production ability of vaccines containing TauRD and FlaB-TauRD recombinant protein according to the number of times of vaccine immunization and the concentration of vaccine.

(8) FIG. 8 shows images depicting that FlaB-TauRD recombinant protein formed aggregates in the form of PHFs.

(9) FIG. 9 shows results confirming the production of a structure recognizing antibody by administration of FlaB-TauRD recombinant protein. The results confirm that an antibody produced by simultaneous reaction of thioflavin S (Th-S), which selectively binds to the beta sheet, and an anti-serum was colocalized while PFH molecules formed a neurofibrillary tangle-like structure.

(10) FIG. 10 shows a tau protein aggregation inhibiting effect by an anti-serum to FlaB-TauRD recombinant protein.

(11) FIG. 11 shows an opsonic phagocytosis stimulating effect of an anti-serum to FlaB-TauRD recombinant protein.

(12) FIG. 12 shows results of expression and purification of norovirus P domain.

(13) FIG. 13 shows expression and purification of Pd-FlaB recombinant protein and specific binding of Pd-FlaB recombinant protein to Pd anti-serum and Flab anti-serum.

(14) FIG. 14 shows results confirming stimulation activity of FlaB and Pd-FlaB to tall-like receptor 5 (TLR5).

(15) FIG. 15 shows an immunization schedule of Pd-FlaB recombinant protein.

(16) FIG. 16 shows results of the introduction of structure recognizing antibody production through protein engineering. Unlike a tau antigen, a structure recognizing antibody was not produced merely when the norovirus Pd antigen was administered in mixing with flagellin (left panel), but a structure recognizing antibody, which did not recognize a monomer only when immunization was conducted using Pd-flagellin fusion antigen and responded to an antigen only on a dot blot experiment using a cell lysate with an antigen structure maintained, was produced (right panel).

(17) FIG. 17 shows an electron microscope observation image of Pd-FlaB recombinant protein.

(18) FIG. 18 shows serum IgG titer by immunization of Pd-FlaB recombinant protein.

(19) FIG. 19 shows serum IgA titer by immunization of Pd-FlaB recombinant protein.

(20) FIG. 20 shows fecal IgG titer by immunization of Pd-FlaB recombinant protein.

DETAILED DESCRIPTION

(21) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Example 1: Production of Alzheimer's Disease Immunization Vaccine

(22) Materials

(23) Culture and Storage of Each Strain

(24) The E. coli strains used in the present invention were incubated in Luria Bertani (LB) medium (Difco Co.). After the incubation, the strains used were stored in an ultralow-temperature refrigerator after glycerol was added to 30%. The strains and plasmids used in the present invention are summarized in Table 1.

(25) TABLE-US-00001 TABLE 1 Strain or plasmid Description Origin Strain DH5α F-φ80dlacZM15(lacZYA-argF)U169deoR recA1 endA1 ATCC hsdR17(rK-mk+) phoA supE44 λ-thi-gryA96 relA1 ER2566 F-λ-fhuA2[lon]ompTlacZ::T7 gene1 gal sulA11(mcrC- New England mrr)114::IS10Rmcr-73::miniTn10-TetS)2R(zgb- Biolabs, Inc. 210::Tn10)(TetS)endA1[dcm] Plasmid pTYB12 N-terminal fusion expression vector, intein tag: Apr being fused New England to N-terminal of target protein Biolabs, Inc.

(26) Methods and Results

(27) 1. Protein Expression and Purification

(28) a. Expression and Purification of Flab Recombinant Protein Derived from Vibrio vulnificus

(29) FlaB recombinant protein was prepared using the genetic sequence (SEQ ID NO: 1) of Flab, which is a flagellar structural component of Vibrio vulnificus CMCP6. In order to obtain a DNA fragment for N-terminal or C-terminal fusion of the flagellin gene flaB, the 1.1 kbp-DNA fragment including flaB gene for N-terminal fusion or C-terminal fusion was amplified using a pair of FlaB-N and FlaB-C primers described in SEQ ID NO: 8 and SEQ ID NO: 9, respectively. That is, PCR reaction using each primer was conducted under conditions of initial denaturation at 95° C. for 5 minutes, 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 60° C. for 30 seconds, and extension at 2° C. for 1 minute, and a final reaction at 2° C. for 10 minutes.

(30) The Intein-CN system by NEB was used as an expression system for the expression of E. coli. The pTYB12 plasmid of the corresponding system was treated with restriction enzymes EcoRI and PstI, and then the amplified flaB PCR product was ligated thereto (pCMM11101). The ligated plasmid was transformed in the E. coli ER2566 expression strain through electric transformation, and only strains living on LB agar plate containing ampicillin, which is a selective marker of the pTYB12 plasmid, were selected, and it was investigated using the PCR primers of SEQ ID NO: 8 and SEQ ID NO: 9 whether the strains contain the corresponding gene product (CMM11101).

(31) The expression of CMM11101 E. coli strain was induced through the addition of 0.5 mM 5-bromoindole-3-chloroisopropyl-D-galactopyranoside (IPTG). The FlaB protein of SEQ ID NO: 2 was obtained from the intein fusion protein using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the instructions of the manufacturer (New England Biolabs Inc.). Endotoxins contained in the isolated protein were removed using AffinityPak™ Detoxi Gel™ endotoxin removing gel (Pierce Inc.).

(32) b. Expression and Purification of Recombinant Tau Repeated Domain

(33) A recombinant protein was prepared from, as an antigen, the whole repeated domain (RD) having high correlation to hyperphosphorylation in the human tau (τ) protein by using E. coli. For the preparation of the recombinant protein, the corresponding gene (SEQ ID NO:04) was subjected to codon optimization for E. coli and gene synthesis (SEQ ID NO: 5). For easy cloning, EcoRI and XhoI restriction enzyme recognition gene sequences were added to the N-terminal and C-terminal, respectively, during gene synthesis. In order to obtain a DNA fragment for fusion, the 1.1 kbp-DNA fragment including tauRD gene for N-terminal fusion or C-terminal fusion was amplified using a pair of tauRD-N primer (SEQ ID NO: 10) and tauRD-C primer (SEQ ID NO: 11). That is, PCR reaction using each primer was conducted under conditions of initial denaturation at 95° C. for 5 minutes, 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 60° C. for 30 seconds, and extension at 72° C. for 1 minute, and a final reaction at 72° C. for 10 minutes. The Intein-CN system by NEB Inc. was used as an expression system for expression of E. coli. The pTYB12 plasmid of the corresponding system was treated with restriction enzymes EcoRI and PstI, and then the amplified tauRD PCR product was ligated thereto (pCMM11102). The ligated plasmid was transformed in the E. coli ER2566 expression strain through electric transformation, and only strains living on LB agar plate containing ampicillin, which is a selective marker of the pTYB12 plasmid, were selected, and it was investigated using PCR primers of SEQ ID NO: 10 and SEQ ID NO: 11 whether the strains contain the corresponding gene product (CMM11102).

(34) The expression of CMM11102 E. coli strain was induced by addition of 0.5 mM 5-bromo-4-indole-3-chloro-isopropyl-D-galactopyranoside (IPTG). The TauRD protein having the amino acid sequence of SEQ ID NO: 3 was obtained from the intein fusion protein by using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the instructions of the manufacturer (New England Biolabs Inc.) Endotoxins contained in the isolated protein were removed using AffinityPak™ Detoxi Gel™ endotoxin removing gel (Pierece Inc.).

(35) For the investigation of the expression of the purified recombinant fusion protein, the molecular weight of the recombinant fusion protein was checked using SDS-PAGE, and as a result, it was verified that a 13 kDa-sized recombinant fusion protein was prepared (FIG. 2).

(36) For the investigation of whether the purified recombinant fusion protein was an accurate tau protein, western blotting was conducted using an anti-tau antibody, and as a result, a band specific to the anti-tau protein was confirmed (FIG. 2).

(37) c. Cloning of Gene for Preparing Recombinant FlaB-TauRD Fusion Protein

(38) The flaB gene of pCMM11101 was treated with EcoRI and PstI restriction enzymes and pCMM11102 was also treated with the same enzymes, and then the flaB gene fragment and the pCMM11102 plasmid were purified through agarose gel electrophoresis. These two genes were ligated to prepare pTYB12::flaB-tauRD gene fusion plasmid (pCMM11103). The ligated plasmid was transformed in the E. coli ER2566 expression strain through electric transformation, and only strains living on LB agar plate containing ampicillin, which is a selective marker of the pTYB12 plasmid, were selected, and it was investigated using the PCR primers of SEQ ID NO: 8 and SEQ ID NO: 11 whether the strains contain the corresponding gene product (CMM11104).

(39) The expression of CMM11103 E. coli strain was induced by addition of 0.5 mM 5-bromoindole-3-chloroisopropyl-D-galactopyranoside (IPTG). FlaB-TauRD fusion protein of SEQ ID NO: 6 was obtained from the intein fusion protein by using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the instructions of the manufacturer (New England Biolabs Inc.) Endotoxins contained in the isolated protein were removed using AffinityPak™ Detoxi Gel™ endotoxin removing gel (Pierece Inc.).

(40) For the confirmation of exactness of the purified FlaB-TauRD, SDS-PAGE and western blotting using a FlaB-specific mouse anti-serum were conducted. As a result, it was verified that the purified FlaB-TauRD fusion protein showed a 57 kDa-sized band having an original size thereof, and bound to the Flab-specific anti-serum on the western blot (FIG. 3).

(41) FIG. 4 shows experimental results that an anti-serum obtained by the immunization of FlaB and Tau-Ag, which is obtained from the expression of a portion of Tau protein, induced the production of a “structure recognizing antibody” responding to a PHF, which is a Tau pathologic conformer. The Tau-Ag used in the immunization was isolated by SDS-PAGE, and then transferred onto a nylon membrane and stained with Ponceau S, and as a result, a monomer protein was confirmed. When a prepared membrane was immuno-blotted with an antibody obtained by the immunization of FlaB and Tau-Ag in the same manner, a monomer band was almost not recognized, and a very small amount of multimer structures, which had not been observed in Ponceau S staining, were strongly recognized. In order to prove this, immunoblotting was conducted after native PAGE while multimeric structures were maintained, and as a result, it was verified that the immune serum strongly responded to the multimeric structures, which have not been recognized by standard serum. This indicates that the anti-serum induced by the present invention shows significantly high binding strength to tau aggregates causing Alzheimer's disease.

(42) d. Characterization of Recombinant FlaB-TauRD Protein

(43) (1) Investigation of TLR5 Stimulating Ability of Recombinant FlaB-TauRD Protein

(44) For the investigation of whether the purified recombinant tau-RD peptide itself retains stimulation ability to TLR5 as an action point of flagellin, 293-T cells were dispensed at 1×10.sup.5 cells per well in a well-plate incubator, and incubated overnight. Then, NF-κ-Luc plasmid (obtained from Prof. Kim Jong-Mok of the Department of Microbiology, Hanyang University), TLR5 gene-cloned P3×Flag-hTLR-5 plasmid (obtained from Steven B. Mizel of the Department of Microbiology and Immunology, Wake Forest University School of Medicine, USA), and β-galactosidase expression control plasmid (Clontech) were simultaneously introduced into the cells by using Effectene (QIAGEN). After additional incubation for 24 hours, the medium was exchanged with a fresh medium. The FlaB and the tau-RD peptide isolated by IMPACT system were treated for a predetermined time, and luciferase activity was measured using a luminescence analyzer (Luminometer, Berthold Inc.) to check the degree of transcription of NF-κB. The results are shown in FIG. 5. In the results of FIG. 5, the recombinant tau-RD peptide used as an antigen did not show TLR5 stimulating ability, but the FlaB-TauRD fusion protein showed significant TLR5 stimulating ability compared with FlaB.

(45) (2) Comparison of Tau Antigen-Specific Antibody Forming Ability According to Administration of Recombinant FlaB-TauRD Mix Vaccine

(46) After six-week-old female Balb/c mice (Orient Bio, Korea) were intranasally immunized with the flagellin tau-RD peptide mix vaccine of the present invention three times, five times, and six times at weekly intervals, serum for each case was obtained to compare the formation of the tau-RD peptide-specific antibody (FIG. 6). For comparison, the anti-serum obtained by coating the recombinant flagellin tau-RD protein on the 96-well plate ELISA plate and conducting immunization was serially diluted two-fold, and checked through indirect ELISA method.

(47) As a result, the flagellin tau-RD peptide mix vaccine showed high serum IgG formation compared with the tau-RD peptide alone immunization group. As for the treatment with antigen at different doses, there was not a statistically significant dose-response relationship in three times immunization, but a statistically significant difference in antigen-specific antibody forming ability was confirmed between the antigen 6 μg treatment group and the antigen 10 μg treatment group in five times immunization. A statistically significant difference in antigen-specific antibody forming ability was not confirmed between the 10 μg treatment group and the 14 μg treatment group (FIG. 7a).

(48) In the comparison results of antigen-specific antibody forming ability after three times, five times, and six times immunization, statistically significant differences in antigen-specific antibody forming ability could be confirmed between the three-time administration groups and the five-time administration groups, but statistically significant differences in antigen-specific antibody forming ability could not be confirmed between the five-time administration groups and the six-time administration groups (FIG. 7b).

(49) (3) Formation of Recombinant FlaB-TauRD Protein Aggregates

(50) It was confirmed through an electron microscope that the anti-serum induced by the present invention, a specific antigen in the brain, self-formed aggregates, and after 5 days of purification, formed aggregates in the form of PHFs.

(51) (4) Induction of Recombinant FlaB-TauRD Protein Aggregates

(52) The tau protein aggregates (aggregates being induced by treatment of purified tau-RD peptide with heparin) was stained with tioflavin S (green), which specifically binds to a β-sheet structure of a protein, and an anti-serum obtained by immunization of a product by the present invention was stained with Alexa fluor 633 (Molecular probe) labeled anti-mouse IgG rabbit IgG, and then the comparison on the presence or absence of the binding between the tau protein and the anti-serum and the degree of the binding was conducted by using a confocal laser microscope.

(53) As a result of the experiment, the anti-serum induced by the present invention showed a binding aspect to the tau protein on the fifth day of aggregation induction (more aggregated) than on the second day of aggregation induction (FIG. 9).

(54) (5) Aggregation Inhibiting Effect of Recombinant FlaB-TauRD Protein

(55) For the investigation of a tau aggregation inhibiting effect of the anti-tau serum induced by the present invention, the recombinant tau-RD peptide was treated with the anti-serum induced by the present invention, followed by anti-serum removal, and then the aggregation of a tau peptide was induced by using heparin, and the degree of aggregate formation was checked through a transmission electron microscope.

(56) The aggregation of tau protein was observed when the tau protein was pre-treated with control serum obtained by immunization of saline, but the aggregation of the tau protein was observed to deteriorate when the tau protein was pre-treated with the anti-serum induced by the present invention (FIG. 10).

(57) (6) Phagocytic Activity of Recombinant FlaB-TauRD Protein

(58) An experiment was conducted to investigate whether the anti-serum induced by the present invention promotes tau aggregate opsonic phagocytosis of microglial cell line (BV2 cell line, dispensed by Professor Moon, Chang-Jong of Chonnam National University Medical School), which are specific antigen-presenting cells in the brain). The aggregation of the recombinant tau-RD peptide was induced for three days by using heparin, followed by staining with FNR 488 (Green, Bioacts, Korea). Tau aggregates were broken by sonication for 10 seconds, and then the incubated BV2 cell line was treated with the tau aggregates together with the anti-tau serum by the present invention. Control serum obtained through PBS immunization was used as a control. After incubation for 30 minutes, the nuclei of BV2 cells were stained with DAPI (blue) and the cell membranes of BV2 cells were stained with wheat germ agglutinin (WGA, red), and then a comparison about the presence or absence of phagocytosis of the broken tau aggregates and the degree of phagocytosis was conducted using a confocal microscope.

(59) As a result, the broken tau aggregates treated with the anti-serum induced by the present invention were phagocytized by BV2 cells, but such a phenomenon was not found in the control serum treatment group. This indicates that the anti-serum induced by the present invention provides high opsonic phagocytosis ability to the specific antigen presenting cells of the brain (FIG. 11).

Example 2: Preparation of Norovirus Immune Vaccine

(60) a. Norovirus P Domain Antigen Sequence and Codon Optimization Thereof

(61) For DNA for an antigen for the preparation of a norovirus vaccine, norovirus P domain-cloned pGEX-4T-1::VAxxx obtained from Cho Kyung-Oh, a professor of Chonnam National University was used. The inserted gene sequence was as described in SEQ ID NO: 12.

(62) b. Cloning of Gene for Preparing Recombinant Pd Antigen

(63) In order to obtain a DNA fragment for fusion of N-terminal or C-terminal of the Pd gene for an antigen, the 1.1 kbp-DNA fragment including Pd gene was amplified by using a pair of Pd—N and Pd—C primers described in SEQ ID NO: 16 and SEQ ID NO: 17 and, as a template, the Pd-containing plasmid of SEQ ID NO: 12. That is, PCR reaction using each primer was conducted under the conditions of initial denaturation at 95° C. for 5 minutes, 30 cycles of denaturation at 95° C. for 30 seconds, annealing at 60° C. for 30 seconds, and extension at 72° C. for 1 minute, and a final reaction at 72° C. for 10 minutes.

(64) The IMPACT-CN system by NEB Inc. was used as an expression system for expression of E. coli. The pTYB12 plasmid of the corresponding system was treated with restriction enzymes EcoRI and PstI, and then the amplified Pd PCR product was ligated thereto (pCMM11105). The ligated plasmid was transformed in the E. coli ER2566 expression strain through electric transformation, and only strains living on LB agar plate containing ampicillin, which is a selective marker of the pTYB12 plasmid, were selected, and it was investigated using PCR primers of SEQ ID NO: 16 and SEQ ID NO: 17 whether the strains contain the corresponding gene product (pCMM11105).

(65) The expression of CMM11105 E. coli strain was induced by addition of 0.5 mM 5-bromoindole-3-chloroisopropyl-D-galactopyranoside (IPTG). The FlaB-Pd fusion protein of SEQ ID NO: 15 was obtained from the intein fusion protein by using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the instructions of the manufacturer (New England Biolabs Inc.) Endotoxins contained in the isolated protein were removed using AffinityPak™ Detoxi Gel™ endotoxin removing gel (Pierece Inc.).

(66) For the investigation of exactness of the purified recombinant Pd protein, SDS-PAGE was conducted (FIG. 12). As a result, the purified recombinant Pd protein showed a 44 kDa-sized band having an original size thereof.

(67) c. Cloning of Gene for Preparing Recombinant FlaB-Pd Fusion Protein

(68) The flaB gene of pCMM11101 was treated with EcoRI and PstI restriction enzymes and pCMM11105 was also treated with the same enzymes, and then the flaB gene fragment and the pCMM11105 plasmid were purified through agarose gel electrophoresis. These two genes were ligated to prepare pTYB12::flaB-Pd gene fusion plasmid (pCMM11106). The ligated plasmid was transformed in the E. coli ER2566 expression strain through electric transformation, and only strain living on LB agar plate containing ampicillin, which is a selective marker of the pTYB12 plasmid, were selected, and it was investigated using PCR primers of SEQ ID NO: 8 and SEQ ID NO: 17 whether the strains contain the corresponding gene product (CMM11106).

(69) The expression of CMM11105 E. coli strain was induced by addition of 0.5 mM 5-bromoindole-3-chloroisopropyl-D-galactopyranoside (IPTG). The FlaB-Pd protein of SEQ ID NO: 15 was obtained from the intein fusion protein by using a chitin bead column and 1,4-dithiothreitol (1,4-DTT) according to the instructions of the manufacturer (New England Biolabs Inc.) Endotoxins contained in the isolated protein were removed using AffinityPak™ Detoxi Gel™ endotoxin removing gel (Pierece Inc.).

(70) For the confirmation of exactness of the purified FlaB-Pd, SDS-PAGE and western blotting using a FlaB- or Pd-specific mouse anti-serum were conducted. As a result, it was verified that the purified FlaB-Pd fusion protein showed a 44 kDa-sized band having an original size thereof, and bound to the Flab- and Pd-specific anti-serum on the western blot (FIG. 13).

(71) d. Characterization of Recombinant FlaB-Pd Protein

(72) (1) TLR5 Stimulating Ability of Recombinant FlaB-Pd Protein

(73) For the investigation of biological activity of FlaB-Pd fusion protein, 293-T cells were dispensed at 1×10.sup.5 cells per well in a 24-well plate incubator, and incubated overnight. Then, NF-κ-Luc plasmid (obtained from Prof. Kim Jong-Mok of the Department of Microbiology, Hanyang University), TLR5 gene-cloned P3×Flag-hTLR-5 plasmid (obtained from Steven B. Mizel of the Department of Microbiology and Immunology, Wake Forest University School of Medicine, USA), and β-galactosidase expression control plasmid (Clontech) were simultaneously introduced into the cells by using Effectene (QIAGEN). After additional incubation for 24 hours, the medium was exchanged with a fresh medium. The FlaB-Pd fusion protein isolated by the IMPACT system was treated for a predetermined time, and luciferase activity was measured using a luminescence analyzer (Luminometer, Berthold Inc.) to check the degree of transcription of NF-κB. The results are shown in FIG. 14.

(74) (2) Recognition of Recombinant FlaB-Pd Protein Structure

(75) For the verification of vaccine efficacy of the prepared Pd and FlaB-Pd proteins, 6-week-old female Balb/c mice purchased from Orient were used.

(76) Following the schedule shown in FIG. 15, Pd (1 μg), FlaB (1.2 μg)+Pd (1.2 μg) mix, and FlaB-Pd (2.2 μg) fusion protein were intranasally administered to mice three times. After 7 days of the last administration, the whole blood of the mice was obtained, and then serum was isolated.

(77) FIG. 16 shows that a structure recognizing antibody was not produced merely when the norovirus Pd antigen was administered in mixing with FlaB, but a structure recognizing antibody, which did not recognize a monomer only when immunization was conducted using recombinant FlaB-Pd protein and responded to an antigen on a dot blot experiment using a cell lysate with an antigen structure maintained, was produced. This indicates that some antigens need to have special antigen structures through protein engineering.

(78) (3) Electron Microscopic Observation

(79) After the recombinant norovirus P domain protein and FlaB-P domain fusion were respectively purified, samples were stained with uranyl acetate, and then 2 μl of each sample was dropped on a carbon grid, followed by drying, and then was observed at an accelerated voltage of 30 kV using JEOL JEM-2100F transmission electron microscope. As a result, the formation of virus-like particle (VLP) structures is observed in the norovirus P domain recombinant protein. In the FlaB-P domain fusion protein, VLP forms were observed while a structure of aggregation of the VLP forms as subunits was observed (FIG. 17).

(80) (4) Observation of IgG and IgA Titers

(81) Following the schedule shown in FIG. 15, a recombinant P domain antigen alone, FlaB and P domain mix, and a FlaB-P domain fusion protein vaccine were administered to 6-week-old female BalB/c mice three through the nasal cavity. After three times immunization, the whole blood (FIG. 18: serum IgG, FIG. 19: serum IgA) and feces (FIG. 20: fecal secretary IgA titer measurement) of the mice were collected, followed by serum isolation, and then an antigen-specific antibody titer using the recombinant P domain protein as an antigen was checked by using ELISA (secondary IgG antibody; Goat Anti-Mouse IgG, Human ads-HRP Cat. No. 1030-05, SouthernBiotech, Birmingham, Ala. 35260, USA: secondary IgA antibody; Goat Anti-Mouse IgA-HRP Cat. No. 1040-05, SouthernBiotech, Birmingham, Ala. 35260, USA) (FIGS. 18 to 20). As a result, it could be verified that the antigen-specific antibody titer was significantly increased in the FlaB+P domain mix administration group and the FlaB-P domain fusion vaccine administration group rather than in the P domain alone administration group.

(82) This application contains references to amino acid sequences and/or nucleic acid sequences which have been submitted herewith as the sequence listing text file. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1.52(e).