KR101957040B1 - Edible vaccine composition containing N-terminal domain of Porcine epidemic diarrhea virus - Google Patents

Abstract

The present invention relates to: a vaccine composition for oral administration for preventing porcine epidemic diarrhea (PED), which contains an N-terminal domain (NTD) of a spike protein isolated from a porcine epidemic diarrhea virus (PEDV) as an active component; and a feed additive containing the same. A purpose of the present invention is to provide the vaccine composition for preventing PED, which allows effective mucosal absorption by oral administration.

Description

A vaccine composition for oral administration containing an N-terminal domain of a porcine epidemic diarrhea virus containing an N-terminal domain of porcine epidemic diarrhea virus

The present invention relates to a method for the treatment of porcine epidemic diarrhea virus (PEDV), which contains the N-terminal domain (NTD) of a spike protein (S protein) isolated from porcine epidemic diarrhea virus PED) and a feed additive comprising the vaccine composition for oral administration.

Porcine epidemic diarrhea virus (PEDV) is the leading cause of epidemic diarrhea in pigs and was first discovered in England in 1971. Infection with PEDV in pigs can lead to large economic losses, since PEDV infection in pigs causes diarrhea, high mortality in young pigs, and weight loss in pigs (adult pigs).

PEDV belongs to the genus AlphaCoronavirus and has a single-stranded positive-sense RNA as a dielectric. The genome of PEDV consists of a total of seven electronic reading frames (ORFs) encoding four structural proteins. The structural proteins include spike glycoprotein (S protein), membrane protein, envelope protein, and nucleocapsid protein. Among the structural proteins, the S glycoprotein is composed of the S1 domain and the S2 domain. The C-terminal domain (CTD) of the S1 domain can bind aminopeptidase N (APN), a receptor involved in binding to the cell membrane of the host cell when PEDV is infected, (BX Li, JW Ge, YJ Li, Virology , 365 (2007) 166-172). It has been reported that the antibody can be used as a major target antigen inducing the production of neutralizing antibodies.

PEDV is mainly infected through a fecaloral route and direct infection of PEDV is observed in the small intestine epithelium. As a result, severe lesions appear in the gastrointestinal tract and the immune response to PEDV begins to be induced around mucosal tissues. Accordingly, it is considered that the oral vaccine is more effective than the subcutaneous injection vaccine for preventing or treating PEDV infection.

However, none of the PEDV vaccines currently available has been developed to be capable of mucosal absorption. This is because the intestinal mucosal tissues have a specific immune system that is different from the systemic immune response, and the PEDV vaccine material currently developed is difficult to cope with the specific immune system of the intestinal mucosa.

The mucosal immune system is composed of an inductive site such as Peyer's patch (PP) in which an antigen-specific immunity induction reaction occurs, and a prolyl layer (lamina propria, LP) producing antigen-specific IgA It has the same immune effector site. Since PP does not have collateral lymph nodes, the main pathway for the entry of the antigen into the PP is through microfold cells (M cells), where the epithelial cells stimulated by the antigen are introduced into the epithelial cells (follicle-associated epithelium). M cells are classified into enterocyte classifications due to their unique morphological features such as thin glycocalyx, short, irregular microvilli, and internal pockets including B cells and antigen-presenting cells (APS) do. Luminal Ag, which has migrated through the interior of M cells, binds to APC and consequently induces secretion of antigen-specific IgA ⁺ B cells. Thus, antigen-specific IgA secreted from plasma B cells of LP is known to form a dimer and serve as a mucosal protector such as immunosuppression, intracellular neutralization and antigen release. Through this series of procedures, a hospital-specific mucosal immune system can be induced to protect host cells against infected pathogens as a result.

Thus, in order to more effectively induce mucosal-specific immunity against the pathogen infections, the importance of a target vaccine material capable of stimulating M cells has emerged. Because M cells are concentrated in epithelial cells of the gastrointestinal tract, direct absorption of these antigenic substances from the target mucosa may affect vaccine efficiency.

Antigens targeted to M cells can promote antigen-to-molecule binding specifically to M cells, and molecules specifically expressed in M cells include GP2 protein, complement 5a receptor, and alpha (1, 2) -fucoose, and the like. For example, a tetanus toxoid conjugated with NKM 16-2-4, an antibody specific for M cells, binds to a carbohydrate moiety containing α (1, 2) -fucose and is targeted It has been reported that oral administration of cholera toxin (CT), which is used as an adjuvant of mucosal absorption vaccine, can induce immunity of systemic immunity and mucosal site significantly There is a bar.

Accordingly, the present inventors have developed a vaccine composition for oral administration to prevent porcine epidemic diarrhea virus (PEDV), and as a result of efforts to enhance the vaccine effect through mucosal absorption, The N-terminal domain (NTD) corresponding to the 501st amino acid sequence specifically stimulates M cells of the mucosa to induce a systemic and mucosal-specific immune system, thereby effectively expressing antigen-specific IgG2a and IgA And confirmed that the N-terminal domain of the present invention can be used as an effective ingredient of a vaccine for oral administration for preventing PEDV infection, thereby completing the present invention.

As described above, the porcine epidemic diarrhea virus (PEDV) is preferably administered orally administered vaccine so that it can be absorbed into the mucous membrane because it is transmitted by oral infection as a main infection route and causes lesions in the gastrointestinal tract However, the vaccine antigens that can effectively induce the mucosal-specific immune system have not been proposed, and research on this has continued.

Accordingly, it is an object of the present invention to provide a vaccine composition for preventing swine diarrhea disease (PED), which can effectively absorb mucous membrane by oral administration.

It is still another object of the present invention to provide a feed composition comprising the vaccine composition.

In order to accomplish the above object, the present invention provides a method for producing a spike protein (S protein), which is isolated from porcine epidemic diarrhea virus (PEDV), comprising an N-terminal domain (NTD) And a vaccine composition for preventing swine-derived diarrhea disease (PED).

The present invention also provides a feed additive comprising the vaccine composition for preventing PED.

In one preferred embodiment of the present invention, the N-terminal domain may be composed of the 231st to 501st amino acid sequence of the S protein consisting of the amino acid sequence of SEQ ID NO: 3. The N-terminal domain may be a protein consisting of the amino acid sequence of SEQ ID NO: 1.

In a preferred embodiment of the present invention, the vaccine composition for preventing PED may be a formulation for oral administration, and may be a formulation which is absorbed into mucosa through mucosal administration.

In a preferred embodiment of the present invention, the vaccine composition for preventing PED may further comprise an immunity enhancer.

Accordingly, the present invention provides a _VED vaccine composition comprising an N-terminal domain (NTD _231-501 protein) corresponding to the 231st to 501st amino acid sequence of the S1 protein of PEDV as an active ingredient. The NTD _231-501 protein of the present invention specifically stimulates M cells in the gastrointestinal mucosa to induce systemic and mucosal-specific immune system, thereby effectively expressing antigen-specific IgG2a and IgA, Of the N-terminal domain (NTD _231-501 protein) can be used as an active ingredient of a vaccine for oral administration for preventing PEDV infection.

Figure 1 is a schematic diagram showing the structure of the N-terminal domain (NTD _231-501 protein) in the PEDV S1 protein. Figure 2 shows the expression process of the NTD _231-501 protein in E. coli :
2A is a vector map of an expression vector for overexpressing the NTD _231-501 protein in E. coli;
Figure 2b shows SDS-PAGE and western blot results to confirm expression levels of NTD _231-501 protein overexpressed in E. coli; And
FIG. 2C shows the process of purifying NTD _231-501 protein overexpressed in E. coli by glutathione sepharose chromatography.
Figure 3 shows the binding potency between NTD _231-501 protein and mucin:
FIG. 3A shows ELISA analysis results for confirming the binding affinity of purified NTD _231-501 protein and mucin; FIG. And
FIG. 3B shows the results of confirming the degree of binding of NTD _231-501 protein to M cells (indicated by a dotted line) in _Feyer 's _patch (PP) tissue.
FIG. 4 shows the result of confirming whether an antigen-specific immune response appears in mice orally administered NTD _231-501 protein:
FIG. 4A shows the results of confirming the level of the systemic immune response by confirming the level of antibody expression in the plasma of mouse NTD _231-501 protein orally; And
4B shows the result of confirming the number of antigen-specific IgA-secreting cells in the proliferation layer (LP) of mice to which NTD _231-501 protein was orally administered.

Hereinafter, the present invention will be described in detail.

As described above, the porcine epidemic diarrhea virus (PEDV) is preferably administered orally administered vaccine so that it can be absorbed into the mucous membrane because it is transmitted by oral infection as a main infection route and causes lesions in the gastrointestinal tract However, the vaccine antigens that can effectively induce the mucosal-specific immune system have not been proposed, and research on this has continued.

Accordingly, the present invention relates to a method for producing a porcine epidemic diarrhea virus (PEDV), which comprises as an active ingredient an N-terminal domain (NTD) of a spike protein (S protein) isolated from porcine epidemic diarrhea virus A vaccine composition for preventing diarrhea (PED) is provided.

In the vaccine composition of the present invention, the S protein is one of structural proteins of PEDV and can be divided into S1 protein and S2 protein. The S1 protein is composed of the 21st to 793th amino acid sequence of the S protein full length consisting of the amino acid sequence of SEQ ID NO: 3, and the S2 protein is composed of the 794th to 1385th amino acid sequence of the S protein total length. Functionally, the S1 protein acts as a domain to which the host cell receptor and the viral protein bind, and the S2 protein plays a role in the insertion of the viral genome into the genome of the host cell. The S1 protein may be further divided into an N-terminal domain (NTD) and a C-terminal domain (CTD). NTD (NTD _231-501 protein) may be composed of the 231st to 501st amino acid sequence of the S protein sequence consisting of the amino acid sequence of SEQ ID NO: 3, and specifically it is preferably composed of the amino acid sequence of SEQ ID NO: 1 . More specifically, the NTD of the present invention is most preferably a protein sequence encoded by a gene consisting of the nucleotide sequence of SEQ ID NO: 2. The NTD can function to exhibit binding ability with sugar residues. In addition, the CTD refers to a portion corresponding to amino acid sequence 502 to 780 of the sequence of the S protein consisting of the amino acid sequence of SEQ ID NO: 3. The CTD corresponds to the pAPN-binding site.

The PED-preventing vaccine composition of the present invention may be administered by a conventional method such as oral administration, intramuscular injection, subcutaneous injection, etc., but is not limited thereto. Specifically, the vaccine composition is preferably formulated into a formulation for oral administration. Formulated as a formulation for oral administration means a route administered to the mucosal membrane in the gastrointestinal tract and is characterized in that the mucosal membrane allows direct absorption of the antigen into the mucosa. From the viewpoint of using the vaccine composition of the present invention as a vaccine for preventing PEDV infection, PEDV induces lesions in the gastrointestinal mucosa through oral infection, and thus it may be effective to activate the mucosal-specific immune system through mucosal administration.

The PED prophylactic vaccine composition of the present invention preferably further comprises an immunostimulant, but is not limited thereto.

In a specific example of the present invention, the inventors of the present invention first used the gene encoding the N-terminal domain (NTD) of the PEDV S1 protein (FIG. 1) to induce overexpression of the NTD _231-501 protein And over-expressed recombinant NTD _231-501 protein (Figure 2).

The present inventors also confirmed that the NTD _231-501 protein binds to specific sugar residues expressed at the end of M cells, as a result of confirming the binding of over-expressed recombinant NTD _231-501 protein and mucin to mucin Confirming that it could function as an antigen capable of targeting M cells when absorbed into the small intestine mucosa (Fig. 3).

In addition, the present inventors _confirmed that the NTD _231-501 protein can bind to M cells and induce both the immunity of the general immunity and the mucosal region. As a result, in the group administered with the NTD _231-501 protein as a vaccine, antigen-specific IgG2a Expression and IgA antibody-secreting cell number (Fig. 4).

Accordingly, the present invention provides a _VED vaccine composition comprising an N-terminal domain (NTD _231-501 protein) corresponding to the 231st to 501st amino acid sequence of the S1 protein of PEDV as an active ingredient. The NTD _231-501 protein of the present invention specifically stimulates M cells in the gastrointestinal mucosa to induce systemic and mucosal-specific immune system, thereby effectively expressing antigen-specific IgG2a and IgA, Of the N-terminal domain (NTD _231-501 protein) can be used as an active ingredient of a vaccine for oral administration for preventing PEDV infection.

Vaccine means a pharmaceutical composition containing at least one immunologically active ingredient that induces an immunological response in an animal. The immunologically active component of the vaccine may contain appropriate elements of a live virus or dead virus (subunit vaccine), whereby these elements destroy the entire virus or its growth culture, and then the desired construct (s) Or by isolation and purification processes induced by appropriate manipulation of a suitable system such as bacteria, insects, mammals or other species, or by appropriate purification of the pharmaceutical composition (Polynucleotide vaccination) by induction of the above synthetic process in an animal in need of vaccination by direct incorporation of the genetic material. The vaccine may comprise one or more of the elements described above.

The vaccine may be in any form known to those skilled in the art, for example, but not limited to, a solid form suitable for the form or suspension of liquids and injections. Such formulations may also be emulsified or encapsulated into liposomes or soluble glass, or may be prepared in the form of aerosols or sprays. These may be contained in transdermal patches. For liquids or injections, they may contain, if necessary, propylene glycol and sodium chloride in an amount sufficient to prevent hemolysis (e.g., about 1%).

The vaccine of the present invention may further comprise a pharmaceutically acceptable carrier or diluent. The term " pharmaceutically acceptable " as used herein refers to a non-toxic composition which is physiologically acceptable and which, when administered to a pig, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .

Suitable carriers for vaccines are known to those skilled in the art and include, but are not limited to, proteins, sugars, and the like. The carrier may be an aqueous solution, or a non-aqueous solution, suspension or emulsion. As immunosuppressants for increasing the immunogenicity, there may be used orthopedic or amorphous organic or inorganic polymers and the like. Immune adjuvants are known to play a role in promoting immune responses through chemical and physical linkage to antigens in general. The immunosuppressants used in this study were atypical aluminum gels, oil emulsions, dual oil emulsions and immunosols. Various plant-derived saponins, levamisole, CpG dinucleotide, RNA, DNA, LPS and various types of cytokines have been used to promote the immune response. Such an immunogenic composition can be used as a composition for inducing an optimal immune response by a combination of various adjuvants and an immune response promoting additive. In addition, as a composition to be added to the vaccine, a stabilizer, an inactivating agent, an antibiotic, a preservative, etc. may be used. Depending on the route of administration of the vaccine, the vaccine antigen may be mixed with distilled water, buffer solution or the like.

The present invention also provides a feed additive comprising the vaccine composition for preventing PED according to the present invention.

Preferably, the feed additive of the present invention is added to the pig feed, but not limited thereto, and can be used without limitation as long as it is used for raising an individual susceptible to infection with PEDV.

The feed additive of the present invention replaces the conventional oral administration vaccine and antibiotic, inhibits the growth of harmful food pathogenic bacteria, improves the health condition of the animal, improves the livestock weight and meat quality, increases the milk yield and immunity It is effective. The feed additive of the present invention can be produced in the form of a fermented feed, a compound feed, a pellet form, a silage reed or the like.

The fermented feed can be prepared by fermenting an organic material by adding various microorganism groups or enzymes other than the vaccine composition for preventing PED of the present invention, and the compounded feed can be prepared by various kinds of general feeds and the vaccine composition for preventing PED of the present invention Can be prepared by mixing. The pellet-shaped feed can be produced by applying heat and pressure to the compound feed or the like in a pelletizer, and the silage can be produced by fermenting a chrysanthemum feed as a microorganism. The wet fermented feed is prepared by collecting and transporting organic matter such as food waste, mixing the excipient and the excipient for moisture control at a certain ratio, fermenting at a suitable temperature for fermentation for more than 24 hours, And the like. The fermented dried feed can be prepared by adjusting the wet fermented feed so that the moisture content is 30% to 40%.

Hereinafter, the present invention will be described in more detail by way of Experimental Examples and Examples. It is to be understood by those skilled in the art that these examples and examples are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these examples and examples .

[Experimental Example]

Hereinafter, an experimental method performed for the embodiment of the present invention will be described. The experimental examples of the present invention can be changed and applied without limitation within the range understood by a general engineer.

Use reagent

All reagents were purchased from Sigma-Aldrich Chemical Co.

BALB / c mice (6 weeks old) were purchased from the Koatech Laboratory Animal Center. All animal experiments were carried out in accordance with the Guidelines approved by the ethics committee on animal breeding and use at Chonbuk National University (CBU 2011-0061 and CBU 2015-0004), and the experiments were conducted according to the guidelines proposed by the committee.

Expression of NTD recombinant protein from PEDV S1 protein

PCR amplification was carried out using a primer set (synthesized by GeneScript) specific for the gene of SEQ ID NO: 2 with the gene encoding the S1 protein of PEDV BM1 as a template and the 231 to 501 amino acids of the NTD domain ) (NTD _231-501 gene) was amplified. The sequences of the primers used are as follows: forward primer; 5'-CGC GGA TCC ACA GCT AAT TGC ATT-3 ', reverse primer; 5'-CCG CTC GAG TCA AAA AGA AAT TGG CTG-3 '. At the time of gene amplification, the gene was designed so that the glutathione S-transferase (GST) tag binds to the N-terminus of the expressed recombinant NTD _231-501 protein. The amplified NTD _231-501 gene was _inserted into pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or pET SUMO vector (Thermo Fisher Scientific, Waltham, VA) to construct the NTD _231-501 protein expression vector (Fig. 2A). The constructed expression vector was transformed with Escherichia coli BL21 (DE3) strain, and the transformant was cultured to induce NTD _231-501 protein expression. After the incubation, the transformants were obtained and the cells were disrupted. NTD ₂₃₁ from the cell lysate was isolated from the cell lysate using glutathione sepharose chromatography or Ni-NTA-based affinity purification system _-501 protein was isolated and purified.

Western Blot

 Western blot was performed to confirm the expression level of the protein.

The NTD _231-501 protein expression-induced transformant-disrupted cell lysate was loaded on a 10% SDS-PAGE gel to separate proteins and transfer proteins to polyvinylidene fluoride (PVDF) membranes. The transferred proteins were immunoblotted using anti-GST antibody (Santa Cruz Biotechnology, Dallas, Tex.) As the primary antibody. In order to visualize the primary antibody bound to GST, a secondary antibody conjugated with horseradish peroxidase (HRP) was added to confirm the expression of GST-tagged NTD _231-501 protein. The degree of expression was confirmed using a chemiluminescence detection kit (GE Healthcare Life Sciences).

Immunofluorescent staining analysis

Peyer's patch (PP) was separated from the small intestine of BALB / c mice to prepare a whole-mount sample. The prepared specimens were treated with 1 μg recombinant NTD _231-501 protein and incubated for 2 hours. The cells were fixed with 4% paraformaldehyde for 48 hours and then incubated with 3% fetal bovine serum albumin (BSA) and 0.1% glycine Lt; / RTI > buffer (PBS). The cells or proteins were then stained by treatment with anti-M cell antibody (NKM 16-2-4), anti-GST antibody or wheat germ agglutinin. The stained cells or proteins were developed using secondary antibodies conjugated with appropriate fluorescent dyes. The degree of color development was analyzed using a confocal laser scanning microscope (LSM 510 META; Carl Zeiss, Oberkochen, Germany).

Mucin-binding assay

Mucin-binding assays of NTD _231-501 recombinant proteins were performed according to previously known methods (X. Li, H. Chen, Appl. Environ Microbiol , 81 (2015) 515-521). First, for ELISA analysis, PBS containing bovine mucin at a concentration of 60 μg / ml was added to a 96-well plate and the PBS was dried to attach the bovine mucin to the bottom of the well plate. The attached mucin was blocked with PBS containing 3% BSA, NTD _231-501 recombinant protein was added to each well at 50 ng per well, and then incubated at 37 ° C for 2 hours. After incubation, plates were washed with PBS and incubated with primary anti-GST antibody (Abcam, Cambridge, UK) and HRP-conjugated secondary antibody was added as secondary antibody. Finally added with 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetramethylbenzidin) substrate solution was measured and then the enzyme activity in a microplate reader, NTD _231-501 recombinant The affinity of the protein with the mucin was confirmed.

ELISA and ELISpot analysis

First, ELISA analysis was performed to determine the level of IgG production in mouse serum. 50 쨉 g of NTD _231-501 recombinant protein was orally administered to female BALB / c mice once a week for a total of 3 weeks. When the recombinant protein was orally administered, it was divided into cholera toxin group and non - cholera toxin group. To determine the NTD-specific serum IgG isotype, four weeks after the last oral administration, serum from mouse blood was isolated and analyzed according to the manufacturer's protocol with ISO-2 (Sigma-Aldrich). The wells were coated with NTD _231-501 recombinant protein and then cultured with mouse serum. After incubation, the wells were washed, and an IgG isotype-specific antibody was added as a primary antibody to induce the reaction. Then, an alkaline phosphatase (AP) -binding antibody was treated as a secondary antibody. Finally, the AP substrate was added and the enzyme activity was confirmed through the degree of color development with a microplate reader, thereby confirming the expression level of IgG in the serum.

In addition, ELISpot analysis was performed to determine the number of NTD-specific IgA-secreting cells in lamina propria (LP). The LP lymphocytes were isolated from the LP of the mice to which the recombinant protein was orally administered by treating with a restriction enzyme solution containing Dnase I (Sigma-Aldrich) and collagenase D (Roche, Basel, Switzerland). The isolated LP lymphocytes were NTD _231-501 recombinant Protein-coated ELISpot plate. Plates were cultured for 24 hours after the addition, and the cells were removed and incubated with AP-conjugated anti-IgA antibody. After incubation, nitro-blue tetrazolium and 5-bromo-4-chloro-3'-indolyphosphate substrate solutions were added to the cells to induce IgA antibody The secreted cells were visualized and the number of cells was confirmed.

Recombinant NTD using the transformant Escherichia coli _231-501 Induction of protein expression

Encoding of the N-terminal domain (NTD) in the PEDV S1 protein was used to prepare a vector for expression of NTD using the gene (Fig. 2A), and overexpression of NTD was induced. The NTD protein used was the 231st to 501st amino acids (SEQ ID NO: 1) of the PEDV S1 protein, and the sequence encoding the protein was the gene sequence consisting of the nucleotide sequence of SEQ ID NO: 2 (FIG.

The NTD was _231-501 protein using E. coli cells induces over-expression, the expression was confirmed in the results of a Western Blot using an anti-antibody -GST, NTD _231-501 protein, the expressed protein was approximately _231-501 NTD And had a molecular weight of 55 kDa (Fig. 2B). The expressed proteins were separated and purified using glutathione sepharose chromatography (Fig. 2C).

NTD _231-501  Confirmation of M-cell interaction of protein and PP

To confirm whether the NTD protein of the 231st to 501st sequence in the PEDV S1 protein is suitable for use as a vaccine antigen. In order to measure an antigen-specific immune response-inducing activity, the binding activity between NTD _231-501 protein and mucin prepared in [Example 1] was confirmed by ELISA analysis. At this time, the control group was _subjected to the same reaction by adding a non-structural 3 protein of a dengue virus known as a mucin-binding protein instead of the NTD _231-501 protein.

As a result, as shown in FIG. 3A, the NTD _231-501 protein showed mucin-binding ability at a significantly higher level than the non-structural 3 protein of the control group, dengue fever virus ( p <0.05). Since the NTD _231-501 protein exhibits sugar-binding ability, NTD _231-501 protein can be used as an antigen of the vaccine to induce an immune response and induce a specific immune response to the PEDV S1 antigen .

In the visceral gut mucosa, M cells are composed of a carbohydrate moiety (alpha (1,2) -fucose-containing carbohydrate moiety) that contains a- (1,2) -fucose that is not expressed in the enterocyte (NKM 16-2-4) (Nochi, Tomonori, et al. Journal of Experimental Medicine 204.12 (2007): 2789-2796), which is known to bind to an antibody specific for M cells (NKM 16-2-4). Thus, the present inventors confirmed whether NTD _231-501 protein can bind to a specific sugar residue expressed in the apical area of M cells. To this end, whole tissue samples of _Feyer 's plate were treated with NTD _231-501 protein and cultured to confirm that specific binding of NTD _231-501 protein to the end of M cells could be formed.

As a result, as shown in Fig. 3B, it was confirmed that the NTD _231-501 protein was located together with the NKM 16-2-4 antibody in the end of M cells in the _Peyer-litter tissue sample (Fig. 3B). Thus, it was _confirmed that NTD _231-501 protein among PEDV S1 can be absorbed into the small intestine mucosa when administered orally, and can function as an antigen capable of targeting M cells.

NTD in systemic immunity and mucosal immunity _231-501 Identification of NTD-specific immune response inducing ability of protein

Antigen targeting M cells can effectively induce an antigen-specific immune response in both systemic and mucosal immunity (Kim, Sae-Hae, and Yong-Suk Jang. Immune network 12.5 2012): 165-175.). In order to ensure that a vaccine antigen for oral administration that is absorbed by the mucosa NTD _231-501 protein may play a role, the present inventors have found that BALB / c mice three weeks NTD ₂₃₁ to the oral administration of NTD _231-501 serum protein produced _Lt ; RTI ID = 0.0 &gt; IgG &lt; / RTI &gt; As a solvent control group, a control group in which only NTD _231-501 protein was orally administered and PBS alone was used.

As a result, as shown in FIG. 4A, it was confirmed that NTD _231-501- specific IgG2a was significantly expressed in serum of the test group orally administered with NTD _231-501 protein. This expression of IgG2a was found to be significantly increased in all experimental groups, regardless of whether they were coadministered with cholorotoxin (CT), which is commonly used as an adjuvant in mucosal absorption vaccines ( p <0.05). The expression of antigen-specific IgG2a is closely related to the induction of Th1-type immune response, which can induce systemic immunity and eliminate the virus in the body.

In addition to systemic immunity, we also confirmed that mucosal site specific immune responses were induced. It was confirmed that an increase in the number of cells secreting NTD _231-501- specific IgA antibody was induced in the LP of the group administered NTD _231-501 protein orally (Fig. 4B). The immunoreactivity of mucosal tissues is known to be related to the expression level of IgA antibody secreted from mucosal tissues, _{confirming that} NTD _231-501 protein can be effectively used as a candidate for vaccine to be absorbed into the mucosa.

Therefore, the NTD _231-501 protein of the present invention can induce an antigen-specific immune response in the mucosal tissues as well as the systemic immune response when administered orally, so that NTD _231-501 protein can be used as a vaccine for oral administration Respectively.

<110> INDUSTRIAL COOPERATION FOUNDATION CHONBUK NATIONAL UNIVERSITY <120> Edible vaccine composition containing N-terminal domain of          Porcine epidemic diarrhea virus <130> 1062862 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 271 <212> PRT <213> Porcine epidemic diarrhea virus <400> 1 Thr Ala Asn Cys Ile Gly Tyr Ala Asp Val Phe Ala Thr Glu Pro   1 5 10 15 Asn Gly His Ile Pro Glu Gly Phe Ser Phe Asn Asn Trp Phe Leu Leu              20 25 30 Ser Asn Asp Ser Thr Leu Val His Gly Lys Val Val Ser Asn Gln Pro          35 40 45 Leu Leu Val Asn Cys Leu Leu Ala Ile Pro Lys Ile Tyr Gly Leu Gly      50 55 60 Gln Phe Phe Ser Phe Asn Gln Thr Ile Asp Gly Val Cys Asn Gly Ala  65 70 75 80 Ala Val Gln Arg Ala Pro Glu Ala Leu Arg Phe Asn Ile Asn Asp Ile                  85 90 95 Ser Val Ile Leu Ala Glu Gly Ser Ile Val Leu His Thr Ala Leu Gly             100 105 110 Thr Asn Phe Ser Phe Val Cys Ser Asn Ser Ser Asn Pro His Leu Ala         115 120 125 Thr Phe Ala Ile Pro Leu Gly Ala Thr Gln Val Pro Tyr Tyr Cys Phe     130 135 140 Phe Lys Val Asp Thr Tyr Asn Ser Thr Val Tyr Lys Phe Leu Ala Val 145 150 155 160 Leu Pro Pro Thr Val Glu Ile Val Ile Thr Lys Tyr Gly Asp Val                 165 170 175 Tyr Val Asn Gly Phe Gly Tyr Leu His Leu Gly Leu Leu Asp Ala Val             180 185 190 Thr Ile Asn Phe Thr Gly His Gly Thr Asp Asp Asp Val Ser Gly Phe         195 200 205 Trp Thr Ile Ala Ser Thr Asn Phe Val Asp Ala Leu Ile Glu Val Gln     210 215 220 Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys Asp Asp Pro Val Ser Gln 225 230 235 240 Leu Lys Cys Ser Gln Val Ala Phe Asp Leu Asp Asp Gly Phe Tyr Thr                 245 250 255 Ile Ser Ser Arg Asn Leu Leu Ser His Glu Gln Pro Ile Ser Phe             260 265 270 <210> 2 <211> 819 <212> DNA <213> Porcine epidemic diarrhea virus <400> 2 ggatccacag ctaattgcat tggttatgct gccaatgtat ttgctactga gcccaatggc 60 cacataccag aaggttttag ttttaataat tggtttcttt tgtccaatga ttccactttg 120 gtgcatggta aggtggtttc caaccaacca ttgttggtca attgtctttt ggccattcct 180 aagatttatg gactaggcca atttttctcc tttaatcaaa cgatcgatgg tgtttgtaat 240 ggagctgctg tgcagcgtgc accagaggct ctgaggttta atattaatga catctctgtc 300 attcttgctg aaggctcaat tgtacttcat actgctttag gaacaaattt ttcttttgtt 360 tgcagtaatt cctcaaatcc tcacttagcc accttcgcca tacctctggg tgctacccaa 420 gtaccttatt attgtttttt taaagtggat acttacaact ccactgttta taaatttttg 480 gctgttttac ctcctaccgt cagggaaatt gtcatcacca agtatggtga tgtttatgtc 540 aatgggtttg gatacttgca tctcggtttg ttggatgctg tcacaattaa tttcactggt 600 catggcactg acgatgatgt ttctggtttt tggaccatag catcgactaa ttttgttgat 660 gcactcatcg aagttcaagg aaccgccatt cagcgtattc tttattgtga tgatcctgtt 720 agccaactca agtgttctca ggttgctttt gaccttgacg atggttttta cactatttct 780 tctagaaacc ttctgagtca tgaacagcca atttctttt 819 <210> 3 <211> 1386 <212> PRT <213> Porcine epidemic diarrhea virus <400> 3 Met Lys Ser Leu Thr Tyr Phe Trp Leu Phe Leu Pro Val Leu Ser Thr   1 5 10 15 Leu Ser Leu Pro Gln Asp Val Thr Arg Cys Ser Ala Asn Thr Asn Phe              20 25 30 Arg Arg Phe Phe Ser Lys Phe Asn Val Gln Ala Pro Ala Val Val Val          35 40 45 Leu Gly Gly Tyr Leu Pro Ile Gly Glu Asn Gln Gly Val Asn Ser Thr      50 55 60 Trp Tyr Cys Ala Gly Gln His Pro Thr Ala Ser Gly Val His Gly Ile  65 70 75 80 Phe Val Ser His Ile Arg Gly Gly His Gly Phe Glu Ile Gly Ile Ser                  85 90 95 Gln Glu Pro Phe Asp Pro Ser Gly Tyr Gln Leu Tyr Leu His Lys Ala             100 105 110 Thr Asn Gly Asn Thr Asn Ala Thr Ala Arg Leu Arg Ile Cys Gln Phe         115 120 125 Pro Ser Ile Lys Thr Leu Gly Pro Thr Ala Asn Asn Asp Val Thr Thr     130 135 140 Gly Arg Asn Cys Leu Phe Asn Lys Ala Ile Pro Ala His Met Ser Glu 145 150 155 160 His Ser Val Val Gly Ile Thr Trp Asp Asn Asp Arg Val Thr Val Phe                 165 170 175 Ser Asp Lys Ile Tyr Tyr Phe Tyr Phe Lys Asn Asp Trp Ser Arg Val             180 185 190 Ala Thr Lys Cys Tyr Asn Ser Gly Gly Cys Ala Met Gln Tyr Val Tyr         195 200 205 Glu Pro Thr Tyr Tyr Met Leu Asn Val Thr Ser Ala Gly Glu Asp Gly     210 215 220 Ile Ser Tyr Gln Pro Cys Thr Ala Asn Cys Ile Gly Tyr Ala Ala Asn 225 230 235 240 Val Phe Ala Thr Glu Pro Asn Gly His Ile Pro Glu Gly Phe Ser Phe                 245 250 255 Asn Asn Trp Phe Leu Leu Ser Asn Asp Ser Thr Leu Val His Gly Lys             260 265 270 Val Val Ser Asn Gln Pro Leu Leu Val Asn Cys Leu Leu Ala Ile Pro         275 280 285 Lys Ile Tyr Gly Leu Gly Gln Phe Phe Ser Phe Asn Gln Thr Ile Asp     290 295 300 Gly Val Cys Asn Gly Ala Val Val Gln Arg Ala Pro Glu Ala Leu Arg 305 310 315 320 Phe Asn Ile Asn Asp Ile Ser Val Ile Leu Ala Glu Gly Ser Ile Val                 325 330 335 Leu His Thr Ala Leu Gly Thr Asn Phe Ser Phe Val Cys Ser Asn Ser             340 345 350 Ser Asn Pro His Leu Ala Thr Phe Ala Ile Pro Leu Gly Ala Thr Gln         355 360 365 Val Pro Tyr Tyr Cys Phe Phe Lys Val Asp Thr Tyr Asn Ser Thr Val     370 375 380 Tyr Lys Phe Leu Ala Val Leu Pro Pro Thr Val Arg Glu Ile Val Ile 385 390 395 400 Thr Lys Tyr Gly Asp Val Tyr Val Asn Gly Phe Gly Tyr Leu His Leu                 405 410 415 Gly Leu Leu Asp Ala Val Thr Ile Asn Phe Thr Gly His Gly Thr Asp             420 425 430 Asp Asp Val Ser Gly Phe Trp Thr Ile Ala Ser Thr Asn Phe Val Asp         435 440 445 Ala Leu Ile Glu Val Gln Gly Thr Ala Ile Gln Arg Ile Leu Tyr Cys     450 455 460 Asp Asp Pro Val Ser Gln Leu Lys Cys Ser Gln Val Ala Phe Asp Leu 465 470 475 480 Asp Asp Gly Phe Tyr Thr Ile Ser Ser Arg Asn Leu Leu Ser His Glu                 485 490 495 Gln Pro Ile Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe             500 505 510 Val Asn Ile Thr Ser Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn         515 520 525 Leu Ile Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val     530 535 540 Asp Thr Arg Gln Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser 545 550 555 560 Tyr Gly Tyr Val Ser Ser Ser Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu                 565 570 575 Gln Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr             580 585 590 Ser Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu         595 600 605 Phe Gly Ser Gly Val Lys Phe Thr Ser Leu Tyr Phe Gln Phe Thr Lys     610 615 620 Gly Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp 625 630 635 640 Val Ser Phe Met Thr Leu Asp Val Cys Thr Lys Tyr Thr Ile Tyr Gly                 645 650 655 Phe Lys Gly Glu Gly Ile Ile Thr Leu Thr Asn Ser Ser Phe Leu Ala             660 665 670 Gly Val Tyr Tyr Thr Ser Asp Ser Gly Gln Leu Leu Ala Phe Lys Asn         675 680 685 Val Thr Ser Gly Ala Val Tyr Ser Val Thr Pro Cys Ser Phe Ser Glu     690 695 700 Gln Ala Ala Tyr Val Asp Asp Asp Val Val Gle Ser Ser Leu 705 710 715 720 Ser Ser Thr Phe Asn Ser Thr Arg Glu Leu Pro Gly Phe Phe Tyr                 725 730 735 His Ser Asn Asp Gly Ser Asn Cys Thr Glu Pro Val Leu Val Tyr Ser             740 745 750 Asn Ile Gly Val Cys Lys Ser Gly Ser Ile Gly Tyr Val Ser Ser Gln         755 760 765 Ser Gly Gln Val Lys Ile Ala Pro Thr Val Thr Gly Asn Ile Ser Ile     770 775 780 Pro Thr Asn Phe Ser Met Ser Ile Arg Thr Glu Tyr Leu Gln Leu Tyr 785 790 795 800 Asn Thr Pro Val Ser Val Asp Cys Ala Thr Tyr Val Cys Asn Gly Asn                 805 810 815 Ser Arg Cys Lys Gln Leu Leu Thr Gln Tyr Thr Ala Ala Cys Lys Thr             820 825 830 Ile Glu Ser Ala Leu Glu Leu Ser Ala Arg Leu Glu Ser Val Glu Val         835 840 845 Asn Ser Met Leu Thr Ile Ser Asp Glu Ala Leu Gln Leu Ala Thr Ile     850 855 860 Ser Ser Phe Asn Gly Asp Gly Tyr Asn Phe Thr Asn Val Leu Gly Val 865 870 875 880 Ser Val Tyr Asp Pro Ala Ser Arg Arg Val Val Gln Lys Arg Ser Phe                 885 890 895 Ile Glu Asp Leu Leu Phe Asn Lys Val Val Thr Asn Gly Leu Gly Thr             900 905 910 Val Asp Glu Asp Tyr Lys Arg Cys Ser Asn Gly Arg Ser Val Ala Asp         915 920 925 Leu Val Cys Ala Gln Tyr Tyr Ser Gly Val Met Val Leu Pro Gly Val     930 935 940 Val Asp Ala Glu Lys Leu His Met Tyr Ser Ala Ser Leu Ile Gly Gly 945 950 955 960 Met Val Leu Gly Gly Phe Thr Ser Ala Ala Leu Pro Phe Ser Tyr                 965 970 975 Ala Val Gln Ala Arg Leu Asn Tyr Leu Ala Leu Gln Thr Asp Val Leu             980 985 990 Gln Arg Asn Gln Gln Leu Leu Ala Glu Ser Phe Asn Ser Ala Ile Gly         995 1000 1005 Asn Ile Thr Ser Ala Phe Glu Ser Val Lys Glu Ala Ile Ser Gln Thr    1010 1015 1020 Ser Lys Gly Leu Asn Thr Val Ala His Ala Leu Thr Lys Val Gln Glu 1025 1030 1035 1040 Val Val Asn Ser Gln Gly Ala Ala Leu Thr Gln Leu Thr Val Gln Leu                1045 1050 1055 Gln His Asn Phe Gln Ala Ile Ser Ser Ile Asp Asp Ile Tyr Ser            1060 1065 1070 Arg Leu Asp Ile Leu Ser Ala Asp Ala Gln Val Asp Arg Leu Ile Thr        1075 1080 1085 Gly Arg Leu Ser Ala Leu Asn Ala Phe Val Ala Gln Thr Leu Thr Lys    1090 1095 1100 Tyr Thr Glu Val Gln Ala Ser Arg Lys Leu Ala Gln Gln Lys Val Asn 1105 1110 1115 1120 Glu Cys Val Lys Ser Gln Ser Gln Arg Tyr Gly Phe Cys Gly Gly Asp                1125 1130 1135 Gly Glu His Ile Phe Ser Leu Val Gln Ala Ala Pro Gln Gly Leu Leu            1140 1145 1150 Phe Leu His Thr Val Leu Val Pro Ser Asp Phe Val Asp Val Ile Ala        1155 1160 1165 Ile Ala Gly Leu Cys Val Asn Asp Glu Ile Ala Leu Thr Leu Arg Glu    1170 1175 1180 Pro Gly Leu Val Leu Phe Thr His Glu Leu Gln Asn His Thr Ala Thr 1185 1190 1195 1200 Glu Tyr Phe Val Ser Ser Arg Arg Met Phe Glu Pro Arg Lys Pro Thr                1205 1210 1215 Val Ser Asp Phe Val Gln Ile Glu Ser Cys Val Val Thr Tyr Val Asn            1220 1225 1230 Leu Thr Arg Asp Gln Leu Pro Asp Val Ile Pro Asp Tyr Ile Asp Val        1235 1240 1245 Asn Lys Thr Leu Tyr Glu Ile Leu Ala Ser Leu Pro Asn Arg Thr Gly    1250 1255 1260 Pro Ser Leu Pro Leu Asp Val Phe Asn Ala Thr Tyr Leu Asn Leu Thr 1265 1270 1275 1280 Gly Glu Ile Ala Asp Leu Glu Gln Arg Ser Glu Ser Leu Arg Asn Thr                1285 1290 1295 Thr Glu Glu Leu Gln Ser Leu Ile Tyr Asn Ile Asn Asn Thr Leu Val            1300 1305 1310 Asp Leu Glu Trp Leu Asn Arg Val Glu Thr Tyr Ile Lys Trp Pro Trp        1315 1320 1325 Trp Val Trp Leu Ile Ile Phe Ile Val Leu Ile Phe Val Val Ser Leu    1330 1335 1340 Leu Val Phe Cys Cys Ile Ser Thr Gly Cys Cys Gly Cys Cys Gly Cys 1345 1350 1355 1360 Cys Cys Ala Cys Phe Ser Gly Cys Cys Arg Gly Pro Arg Leu Gln Pro                1365 1370 1375 Tyr Glu Val Phe Glu Lys Val His Val Gln            1380 1385

Claims (7)

(N-terminal domain, NTD) of a spike protein (S protein) isolated from porcine epidemic diarrhea virus (PEDV) as an active ingredient, (PED) comprising the amino acid sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 3.
delete The vaccine composition for preventing PED according to claim 1, wherein the N-terminal domain is a protein consisting of the amino acid sequence of SEQ ID NO: 1.
The vaccine composition for preventing PED according to claim 1, wherein the vaccine composition for preventing PED is a formulation for oral administration.
The vaccine composition for preventing PED according to claim 1, wherein the vaccine composition for preventing PED is absorbed into mucosa through mucosal administration.
The vaccine composition for preventing PED according to claim 1, wherein the vaccine composition for preventing PED further comprises an immunity enhancer.
A feed additive comprising the vaccine composition for preventing PED according to any one of claims 1, 3, 4, 5 and 6.

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