KR101919002B1 - Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof - Google Patents

Abstract

In the present invention, the major antigenic epitopes in the VP1 protein of foot-and-mouth disease viruses having different serotypes are codon-optimized, and then a fusion protein antigen (Multi-VP1 epitopes) is converted into a soluble protein The present invention relates to a vector capable of expressing or purifying an Escherichia coli, a vector transformed thereby, a soluble Multi-VP1 epitope protein expressed and purified from the microorganism, and a vaccine for the protein. More particularly, An Escherichia coli expression vector containing an O, A, Asia1 type foot-and-mouth disease virus or a gene linking major antigen epitopes in the VP1 protein of different local foot-and-mouth disease viruses in the same serotype, a specific recombinant Escherichia coli And a soluble Multi-VP1 epit < RTI ID = 0.0 > epitope < / RTI & ope protein as an active ingredient.

Description

Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof "

The present invention relates to a fusion protein comprising major antigenic epitopes in the VP1 protein of foot-and-mouth disease viruses having different serotypes. More particularly, the present invention relates to a fusion protein comprising a fusion protein antigen (Multi-VP1 epitopes) A recombinant microorganism transformed with the vector, and a soluble fusion protein antigen obtained from the recombinant microorganism as an active ingredient. The present invention also relates to a vaccine for preventing foot-and-mouth disease.

The Foot and Mouth Disease virus is a single stranded bipolar RNA virus that is deposited in the capsid composed of structural proteins without the envelope of viruses belonging to the genus Picornaviridae and Aphthovirus. There are seven serotypes, A, O, C, Asia1, SAT1, SAT2 and SAT3. The major serotypes are divided into more than 200 subtypes and vary in serotype or serotype. Crossing immunity is not achieved, so prevention and prevention of diseases are very difficult diseases.

Currently, foot-and-mouth disease vaccines are in widespread use worldwide for inactivated vaccines. However, there is a danger that the vaccine for inactivating foot-and-mouth disease is made by using foot-and-mouth disease pathogens. There is a possibility that the vaccination may result in a slight increase of the virus in the epithelial cells, so that there is a possibility that the vaccinated livestock remains carrier to the actual foot-and-mouth disease virus in the future. Second, on the extension of the problem of direct use of foot-and-mouth disease virus, existing vaccines have disadvantages in that they require facilities with high level of protection in the manufacturing process. Since viruses used in laboratories or vaccine manufacturing factories can spread out and act as infectious agents, they can be a problem in itself, and it is difficult to easily manufacture vaccines in cleaner countries where foot-and-mouth disease does not occur. Third, there is a drawback that it is difficult to verify the efficacy or risk of the vaccine. That is, animal testing in a shielded facility is always required for risk assessment, and it is difficult to keep the performance constant for each vaccine manufactured. Lastly, existing vaccines contain large amounts of non-structural proteins because they make up the supernatant of the cells infected with foot-and-mouth disease virus, and in fact, the NSP, which distinguishes between animals infected with FMD virus and animals immunized with vaccination -antibody testing can be difficult. Therefore, it is necessary to develop a next generation vaccine against foot-and-mouth disease virus. Since the vaccine does not make use of the whole foot-and-mouth disease virus, special facilities are not necessary and a uniform performance vaccine can be manufactured. It should proceed in the direction that it can.

Foot-and-mouth disease viruses are difficult to produce vaccines because of their genetic complexity and diversity, as well as the many variants between serotypes and the presence of many strains within one serotype. In addition, the vaccine against one serotype may not have protective immunity against other serotypes, and in some cases there may be a difference of up to 30% of the total genes in gene sequences of two strains belonging to the same serotype. It should be applied to each type. Therefore, it is important to identify the viral serotypes that are prevalent in the outbreak area of foot-and-mouth disease vaccines and to quickly prepare vaccines based on their serotypes. To this end, it is necessary to improve the current foot-and-mouth disease inactivation vaccine development and production methods. Therefore, it is necessary to develop other types of vaccines that can complement the problems of existing inactivated vaccines and highlight their advantages. In other words, it is necessary to develop a foot-and-mouth disease vaccine capable of rapid reproduction according to the serotypes, a special facility-free and uniformly manufactured vaccine.

To date, various studies for the next generation vaccines have been carried out to overcome the shortcomings of existing inactivated vaccines. For example, vaccines using foot-and-mouth disease virus recombinant virus, VP1 purified from viral particles (Challa et al., Vaccine 25: 3328-3337, 2007), VP1 made by biotechnology, chemically synthesized VP1 peptides (Zhang et al. Virology journal 8: 268, 2011), VP1 peptide epitopes (Gao et al, Virologica Sinica 25 :. 18-26, 2010), viral vector expressing the VP1 epitope, bacterial vector, plant vector (Andrianova et al, Biochemistry 76. : (Wang et al., Vaccine 26: 5135-5144, 2008), VP1-VP4 capsid delivery via replication defective human adenoviral vectors or delivery of the VP1 epitope (Lu et al., Vaccine 26: 48-53, 2008). Among the next-generation vaccines that have been developed, there have been efforts to mass-produce and purify the major epitope of foot-and-mouth antigen in recombinant E. coli as a vaccine (Xin et al., African Journal of Microbiology Research 5 : 486-495, 2011). However, development of recombinant foot-and-mouth disease vaccine has been limited to date due to the difficulty of purification and mass production due to the expression of insoluble protein in E. coli expression.

 Challa, S. et al. Non-toxic Pseudomonas aeruginosa exotoxin A expressing the FMDV VP1 G-H loop for mucosal vaccination of swine against foot and mouth disease virus. Vaccine 25, 3328-3337, doi: 10.1016 / J.Vaccine.2007.01.006 (2007).

Accordingly, the present inventors have conducted studies to express a large amount of Multi-VP1e antigen protein as a basic step for the development of a recombinant foot-and-mouth disease vaccine, and found that a codon-modified form of Multi-VP1e antigen protein is not aggregated in a specific host cell And showed high solubility, and that the commercialization vaccine and antibody production ability were similar to each other. Thus, the present invention was completed.

Accordingly, an object of the present invention is to provide a Multi-VP1e antigen protein capable of inducing a strong immune response against various foot-and-mouth disease virus subtypes.

Another object of the present invention is to provide a polynucleotide encoding the Multi-VP1e antigen protein.

Another object of the present invention is to provide a recombinant vector comprising said polynucleotide.

It is another object of the present invention to provide an antigen expression system capable of expressing the Multi-VP1e antigen protein in the form of Soluble protein in a large amount and purifying the same.

It is still another object of the present invention to provide a vaccine for preventing foot-and-mouth virus infection comprising the Multi-VP1e antigen protein as an active ingredient.

In order to accomplish the above object, the present invention provides a soluble Multi-Vp1e antigen protein comprising a plurality of epitopes according to serotype of foot-and-mouth disease virus (FMDV) VP1 protein.

The present invention also provides a polynucleotide encoding said soluble Multi-VP1e antigen protein.

The present invention also provides a recombinant vector comprising said polynucleotide.

The present invention also provides an antigen expression system capable of expressing the Multi-VP1e antigen protein in the form of Soluble protein in a large amount and purifying the same.

The present invention also provides a method for mass-producing Multi-VP1e antigen protein using the antigen expression system.

The present invention also provides a foot-and-mouth disease vaccine composition comprising the soluble Multi-VP1e antigen protein as an active ingredient.

The present invention also provides a method of enhancing immunity against foot-and-mouth disease virus, comprising administering said soluble Multi-VP1e antigen protein to a breast.

The Multi-VP1e antigen protein according to the present invention induces an excellent immune response to various types of foot-and-mouth disease virus subtypes.

In addition, since it is not made using the whole foot-and-mouth disease virus, the antigen can be manufactured in a general laboratory facility rather than a special facility, and the antigen can be safely supplied because there is no concern about infectivity.

The recombinant microorganism according to the present invention can mass-produce the Multi-VP1e antigen protein in the form of a soluble protein, and it is possible to replace antigens expressed in a short period of time against economical and anticipated foot-and-mouth disease virus serotypes, .

Figure 1 shows the amino acid sequences of two epitopes present in the FM1 O1 / Manisa, A / Pocheon, and Asia / Shamir VP1 proteins.
Fig. 2 shows the expression vectors of the multi-VP1e antigen protein of foot-and-mouth disease virus O1 / Manisa, A / Pocheon, and Asia / Shamir.
Figure 3 shows the peptide sequences of O1 / Manisa, A / Pocheon, and Asia / Shamir used for antibody production and ELISA.
FIG. 4 shows the purified FMDV Multi-VP1e antigen protein by Western blot using Coomassie brilliant blue staining method and each antibody obtained using the peptide of FIG.
FIG. 5 is a graph showing the results obtained by mixing a mouse Multi-VP1e antigen protein and ISA201 from SEPPIC, a commercial preparation vaccine, ISA201 alone, and then collecting serum for confirmation of antibody production and after the administration of footbath virus ASIA1 Shamir strain It is a schematic diagram of the schedule.
FIG. 6 is a diagram showing the Multi-VP1e (O1 / Manisa, A / Pocheon, Asia / Shamir) administration group of the mouse experiment proceeding constantly in FIG.
FIG. 7 shows the results obtained by collecting sera according to the schedule shown in FIG. 5 in the experimental group administered with the Multi-VP1e antigen protein and the mixed preparation of ISA201, the positive control group administered with the commercial vaccine and the negative control group administered with ISA201 alone, It was confirmed by dilution magnification that antibody production in mouse serum was achieved at a high rate through ELISA performed by coating virus VP1 peptide.
FIG. 8 shows the results of the SP ELISA performed by coating inactivated foot-and-mouth disease virus on each type of antibody in the serum used in FIG. 7.
FIG. 9 is a graph showing a high survival rate of a group administered with a Multi-VP1e antigen protein mixed preparation against a foot-and-mouth disease ASIA1 Shamir strain infection after administration of a Multi-VP1e antigen protein mixture preparation, a commercialization vaccine, ISA201, Fig.
FIG. 10 shows the results of qRT-PCR for the complete removal of virus from day 1 in the group administered with the Multi-VP1e antigen protein mixed preparation after the foot-and-mouth disease ASIA1 Shamir strain infection of FIG.
FIG. 11 shows activation of T-immunoreactive cells by administration of Multi-VP1e antigen protein through ELISPOT performed by stimulating spleen cells isolated from each mouse group with Multi-VP1e antigen protein and VP1 peptide shown in FIG. 2 It is the figure which confirms that it is lost.
FIG. 12 is a diagram showing results of screening pigs to be used in the experiment by using PrioCHECK FMDV SP ELISA.
FIG. 13 is a graph showing the results of an 8-week-old pig being fed on a farm by SP ELISA. Serum was collected from each experimental group administered with a multi-VP1e antigen protein mixed preparation and a control group administered with ISA to confirm antibody production It is a schematic diagram of the experiment schedule.
FIG. 14 is a diagram showing a multi-VP1e (O1 / Manisa, A / Pocheon, Asia Shamir) administration group of pigs proceeding according to the schedule of FIG.
FIG. 15 is a graph showing the results of ELISA in which sera of pigs collected by proceeding as shown in FIG. 13 was diluted by the ratio and coated with each type of foot-and-mouth disease virus VP1 peptide, And the rate of formation of the pigs was higher than that of pigs.
FIG. 16 is a graph showing the results of SP ELISA for each type of antibody produced in the serum of pigs collected in the same manner as in FIG. 13.
Fig. 17 is a diagram showing the result of screening of pigs for verifying the efficacy of Multi-VP1e antigen protein. Fig.
FIG. 18 is a schematic diagram of the schedule of the multi-VP1e antigen protein mixed preparation and the commercialization vaccine and the schedule of the foot-and-mouth disease virus ASIA1 Shamir strain infection after the administration of ISA201 in pigs selected by SP ELISA on 8-week-old pigs raised on the farm.
FIG. 19 is a diagram showing changes in body temperature due to foot-and-mouth disease virus infection of a multi-VP1e (O1 / Manisa, A / Pocheon, Asia Shamir)
FIG. 20 is a graph showing the clinical symptoms of each group in the case of the foot-and-mouth disease virus ASIA1 Shamir strain infection, which was progressed as shown in FIG. 18, and the group administered with the multi-VP1e antigen protein- It is also confirmed.
21 shows amino acid sequences of antigenic sites of foot-and-mouth disease virus O / Jincheon, A / Pocheon, and Asia / Shamir.
FIG. 22 is a schematic diagram showing recombination into a pHis parallel vector for the synthesis of a multi-VP1e antigen protein expressing the VP1 epitope of O / Jincheon, O / Pocheon, and ASIA1 / Sharmir, which are serotypes of ongoing foot-and-mouth disease virus.
FIG. 23 is a view showing the Multi-VP1e antigen protein purified using the gene of FIG. 21 by Coomassie brilliant blue staining and Western blotting.
FIG. 24 is a diagram showing Multi-VP1e (O / Jincheon, A / Pocheon, Asia / Shamir) administration group of the mouse experiment proceeding constantly in FIG.
FIG. 25 shows the results of ELISA in which sera were collected from mice administered with the purified Multi-VP1e antigen protein of FIG. 5 and coated with each type of foot-and-mouth virus VP1 peptide, The results are shown in FIG.
Fig. 26 is a chart showing antibody production in the mouse serum of Fig. 25 measured using SP ELISA for each type of foot-and-mouth disease virus.
FIG. 27 shows that a group administered with purified Multi-VP1e antigen protein and a commercialized vaccine, a group administered with Multi-VP1e antigen protein against a foot-and-mouth disease ASIA1 Shamir strain infection in mice administered with ISA201 showed a high survival rate.
FIG. 28 is a graph showing activation of T-immunoreactive cells by administration of Multi-VP1e antigen protein using the serotype of foot-and-mouth disease virus that is currently in vogue using ELISPOT after separating mouse spleen cells from each group.
29 is a diagram showing amino acid sequences of antigenic sites of foot-and-mouth disease virus O / Jincheon, O / Andong, and O / Manisa.
FIG. 30 is a schematic diagram showing recombination into a pHis parallel vector for the synthesis of Multi-VP1e antigen protein expressing VP1 epitope of foot-and-mouth disease virus O / Jincheon, O / Andong and O / Manisa.
31 is a graph showing the results of immunoblot analysis of the peptide used in the ELISA for the preparation of antibodies used for identification of purified FMD virus O / Jincheon, O / Andong, O / Manisa Multi-VP1e antigen protein on SDS- ≪ / RTI > for each type.
Figure 32 shows the results of confirming the Multi-VP1e antigen protein of purified foot-and-mouth disease virus O / Jincheon, O / Andong, O / Manisa by Coomassie brilliant blue staining and Western blotting using each antibody obtained using the peptide of Figure 31 .
FIG. 33 is a graph showing the results obtained by collecting sera from mice administered with the Multi-VP1e antigen protein of foot-and-mouth disease virus O / Jincheon, O / Andong, and O / Manisa as shown in FIG. 5 and then coating each type of foot- The results of the ELISA showed that a high ratio of antibody production in the mouse serum was achieved by dilution magnification.
FIG. 34 shows the activation of T-immunoreactive cells by administration of Multi-VP1e antigen protein of foot-and-mouth disease virus O / Jincheon, O / Andong, and O / Manisa using ELISPOT after separating mouse spleen cells from each group .
35 shows amino acid sequences of antigenic sites of foot-and-mouth viruses A / Pocheon, A / Malay97 and A / Iraq.
FIG. 36 is a schematic diagram showing recombination of a pHis parallel vector for the synthesis of Multi-VP1e antigen protein expressing the VP1 epitope of foot-and-mouth viruses A / Pocheon, A / Malay97 and A /
37 is a graph showing the results of immunoblot analysis of the peptides used in the ELISA for the preparation of antibodies used to identify the purified FMD viruses A / Pocheon, A / Malay97 and A / Iraq Multi-VP1e antigen proteins on SDS- ≪ / RTI > for each type.
FIG. 38 shows the results of Western blot analysis using purified anti-foot-and-mouth virus A / Pocheon, A / Malay97, and A / Iraq Multi-VP1e antigen protein using Coomassie brilliant blue staining method and each antibody obtained using the peptide of FIG. 37 .
FIG. 39 is a graph showing the results obtained by collecting sera from mice administered with the Multi-VP1e antigen protein of foot-and-mouth disease virus A / Pocheon, A / Malay97 and A / Iraq as shown in FIG. 5 and then coating each type of foot- The results of the ELISA showed that a high ratio of antibody production in the mouse serum was achieved by dilution magnification.
40 shows the activation of T-immunoreactive cells by administration of Multi-VP1e antigen protein of foot-and-mouth viruses A / Pocheon, A / Malay97 and A / Iraq using ELISPOT after separating mouse spleen cells of each group .
41 is a diagram showing amino acid sequences of antigenic sites of foot-and-mouth disease viruses Asia / Shamir, Asia / Mongolia 05, and Asia / LC04.
FIG. 42 is a schematic diagram showing recombination of pHis parallel vector for the synthesis of Multi-VP1e antigen protein expressing VP1 epitope of foot-and-mouth disease virus Asia / Shamir, Asia / Mongolia 05 and Asia / LC04.
Figure 43 is a graph showing the results of immunohistochemical analysis of the peptides used in the ELISA for the preparation of antibodies used to identify the purified FMD viruses Asia / Shamir, Asia / Mongolia 05, Asia / LC04 on SDS- ≪ / RTI > for each type.
Figure 44 shows the results of Western blot analysis of the Multi-VP1e antigen protein of purified FMD viruses Asia / Shamir, Asia / Mongolia 05, Asia / LC04 using Coomassie Brilliant Blue staining method and each antibody obtained using the peptide of Figure 43 .
FIG. 45 is a graph showing the results obtained by collecting sera from mice administered with the Multi-VP1e antigen protein of foot-and-mouth disease virus Asia / Shamir, Asia / Mongolia 05, and Asia / LC04 as shown in FIG. 5 and then coating each type of foot- The results of the ELISA showed that a high ratio of antibody production in the mouse serum was achieved by dilution magnification.
46 shows the activation of T-immunoreactive cells by administration of Multi-VP1e antigen protein of foot-and-mouth disease virus Asia / Shamir, Asia / Mongolia 05 and Asia / LC04 using ELISPOT after separating mouse spleen cells of each group .

The present invention provides a Multi-Vp1e antigen protein in which a plurality of epitopes of the VP1 protein according to the foot-and-mouth disease virus (FMDV) serotype are simultaneously expressed.

In the present invention, the Multi-Vp1e antigen protein can have RGD motifs and C-terminus epitopes according to three serotypes randomly arranged. For example, the Multi-VP1e antigen protein of foot-and-mouth disease virus can be composed of the RGD motif and the C-terminus of foot-and-mouth disease virus VP1 protein in the form as shown in FIG. 1, but the O1 / Manisa, A / Pocheon, As shown in FIG. 21, the present invention is not limited to specific serotypes of foot-and-mouth disease viruses but includes all the functional equivalents applied to modified forms including the VP1 epitope for O / Jincheon, A / Pocheon and Asia / Shamir, .

The term " functional equivalent " as used herein refers to a purified antigen protein comprising the VP1 epitope for various types of foot-and-mouth disease viruses, which comprises at least one serotype VP1 epitope, preferably three serotypes, Quot; means various forms of purified antigen protein that can be synthesized and used for the above serotype.

In the present invention, the antigen epitope of the FMDV VP1 protein useful for the construction of the recombinant protein is the amino acid residues 132-162 and 192-212 of the FMDV VP1 protein (SEQ ID NOS: 1-6, SEQ ID NOS: 9-14, To 22 and SEQ ID NOS: 33 to 40). However, the present invention is not limited thereto.

In other words, the Multi-VP1e antigen protein of the present invention includes various kinds of VP1 epitopes showing immunity in the host in addition to the RGD motif and the C-terminus, and includes variants corresponding to each type of protein mutation according to the prevalence of the virus. The term " variant " in the present invention means that one or more amino acid residues are deleted, inserted, non-conserved or conservatively substituted or complexed with the amino acid sequence of the epitope of the VP1 protein in which a particular serotype is expressed, do.

In a preferred embodiment of the antigenic protein of the invention, said randomly arranged epitope of the recombinant Multi-VP1e antigen protein is linked to each other via a peptide linker. Peptide linkers between antigen epitopes should not affect the linear structure of the antigen epitope. Peptide linkers that can be used in the present invention are, for example, GPGPG (SEQ ID NO: 41), GGGPGGGGP (SEQ ID NO: 42) and GGGGP (SEQ ID NO: 43), preferably GPGPG. The experimental data provided herein demonstrate that the " GP linker " does not affect the linear structure of antigen epitopes randomly arranged in the recombinant Multi-VP1e protein in the present invention, and that the recombinant Multi-VP1e protein induces the production of protective antibodies in the animal Lt; / RTI > can be used as a possible peptide linker.

In the present invention, the Multi-VP1e antigen protein may have an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24 and 40, but is not limited thereto.

In the present invention, the Multi-VP1e antigen protein can induce a humoral immune response or a cellular immune response in a target animal.

According to another embodiment of the present invention, the present invention relates to a polynucleotide encoding said Multi-VP1e antigen protein.

Methods for obtaining polynucleotides encoding the Multi-VP1e antigen protein of the present invention are known in the art. For example, polynucleotide molecules can be prepared directly through chemical synthesis. Alternatively, first, each nucleic acid molecule encoding various antigenic epitopes according to the serotypes to be protected is obtained, and the sequences encoding the peptide linkers are added, and then these are ligated together to encode the entire Multi-VP1e antigen protein It is possible to form a polynucleotide.

According to another embodiment of the present invention, the polynucleotide is modified in accordance with the codon usage of the E. coli. When the polynucleotide of the recombinant antigen is composed of a major codon frequently used in Escherichia coli and expressed in Escherichia coli , the expression probability and the expression level of the soluble protein are remarkably increased (Sorensen, MA, Kurland, CG & Pedersen , Journal of molecular biology 207, 365-377, 1989, Zhang, SP, Zubay, G. & Goldman, Gene 105, 61-72 1991, Rosano, GL & Ceccarelli, EA Microbial cell Factories 8, 41, 2009), as well as problems that may arise during protein expression using polynucleotides of minor codons that are less frequently used. That is, if a protein is expressed using a codon having a low frequency of use in E. coli, the translation may be interrupted or the nucleotide sequence may be changed (frame shifting) and the amino acid may be mis- Proteins may not be expressed (Robinson, M. et al. Nucleic acids research 12, 6663-6671, 1984). When a codon having a low frequency of use is used in Escherichia coli, Escherichia coli affect the growth coli) may cause problems in the protein expression level (V Emilsson & CG Kurland EMBO Journal 9 (13) , 4359-4366, 1990).

The codon-modified polynucleotide of the present invention may comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 7, 15, 23 and 31.

According to another embodiment of the present invention, the present invention relates to a recombinant vector comprising said polynucleotide. Expression vectors, e. G. Prokaryotic or eukaryotic expression vectors, which can be used to introduce polynucleotides of the invention into host cells are known in the art.

According to another embodiment of the present invention, the present invention relates to a recombinant microorganism transformed with said vector. Since the expression amount and the expression of the target protein are different depending on the recombinant microorganism (host cell), it is important to select the most suitable host cell for the purpose of mass production of the protein.

The recombinant microorganism (host cell) that can be used to express the Multi-VP1e antigen protein of the present invention is preferably Escherichia It can be coli). Examples of such E. coli strains include BL21-Codon Plus (DE3) -RIPL, BL21-Codon Plus (DE3) -RG, OverExpress ™ C41 (DE3) and C43 (DE3) Competent Cells, Value 10 ⁷ HIT-21 , DH5? Competent Cells, and the like, preferably BL21-Codon Plus (DE3) -RIPL.

(DE3) -RIPL, BL21-Codon Plus (DE3) -RG, OverExpress ™ (DE3) -RG, and the like to find an E. coli host cell system in which the codon-modified Multi-VP1e antigen protein is expressed in a form having high solubility C41 (DE3) and C43 (DE3) Competent Cells, Value 10 ⁷ HIT-21 and DH5α Competent Cells were tested by introducing various codon-modified polynucleotides. In the case of OverExpress ™ C41 (DE3) and C43 (DE3) Competent Cells and Value 10 ⁷ HIT-21 and DH5α ™ Competent Cells, the Multi-VP1e antigen protein was not expressed in water-soluble form and BL21-Codon Plus DE3) -RG cells were expressed in a small amount of water-soluble Multi-VP1e antigen protein, but they were expressed at a yield 10 times lower than that of BL21-Codon Plus (DE3) -RIPL cells and finally BL21-Codon Plus (DE3) -RIPL cells express the soluble protein with the highest yield, and completed the present invention.

These results suggest that the BL21-Codon Plus (DE3) -RIPL cells can express the Multi-VP1e antigen protein more efficiently by including the gene that expresses the tRNA further. In addition, BL21-Codon Plus (DE3) -RIPL cells express genes such as argU, ileY and LeuW as well as tRNA genes, which facilitates protein expression of the AT- or GC-rich genome, And the like.

According to another embodiment of the present invention, the present invention provides a method for mass-producing soluble Multi-VP1e antigen protein comprising culturing the recombinant microorganism. Specifically, the mass production method of the soluble Multi-VP1e antigen protein

(a) preparing a recombinant vector comprising a polynucleotide encoding a Multi-VP1e antigen protein;

(b) transforming and culturing Escherichia coli with the vector;

(c) digesting the E. coli cultured in step (b) to obtain a Multi-VP1e antigen protein, and then mass-producing the soluble Multi-VP1e antigen protein.

The temperature for inducing the expression of the Multi-VP1e protein from the transformed microorganism cultured in the step (b) may be in the range of 15 ° C to 25 ° C, and most preferably 18 ° C to 22 ° C. As shown in step (b), when the expression of Multi-VP1e antigen protein is induced at a specific temperature, the amount of Multi-VP1e antigen protein having high solubility is increased.

(DE3) -RG, OverExpressTM C41 (DE3) and C43 (DE3) Competent Cells, Value 10 ⁷ HIT-21 transformed with a recombinant vector encoding a Multi-VP1e antigen protein in a preliminary experiment , DH5α ™ Competent Cells and BL21-Codon Plus (DE3) -RIPL were subjected to protein expression at 37 ° C. and 20 ° C., respectively, and the expression and solubility of the Multi-VP1e antigen protein was determined by electrophoresis Respectively. As a result, it was confirmed that the Multi-VP1e antigen protein expressed at 20 ° C had higher solubility than the protein expressed at 37 ° C.

According to another embodiment of the present invention, the present invention relates to a vaccine composition for preventing and treating FMD virus comprising the Multi-VP1e antigen protein as an active ingredient.

In the present invention, the vaccine composition may further comprise a pharmaceutically acceptable carrier and / or adjuvant. Suitable carriers and / or adjuvants include, for example, aluminum hydroxide or aluminum phosphate as an inorganic adjuvant; Organic adjuvants such as CpG DNA or poly A; Microorganisms and their extracts; And oil adjuvants, and the adjuvant used in the present invention is not limited to ISA201 or any particular kind of adjuvant.

According to another embodiment of the present invention, the present invention provides a method for enhancing immunity against foot-and-mouth disease virus in an animal, comprising administering an effective amount of said Multi-VP1e antigen protein or vaccine composition to an animal in need thereof .

The vaccine of the present invention may be administered by any route suitable for administration of a protein vaccine, for example, by subcutaneous, intramuscular, intraperitoneal or intravenous injection, preferably to the animal twice a day Can be administered by intramuscular injection.

The term " effective amount " as used herein means that the component of the vaccine is in an amount sufficient to induce an immune response specific for FMDV in the vaccinated animal. Effective amounts can be readily determined by those skilled in the art and can be determined, for example, through routine experimentation in animals. The protein component of the vaccine of the present invention may be administered in a total amount of 100 to 2000 μg, preferably 200 to 1000 μg.

In a specific embodiment of the present invention we have identified two epitopes of VP1 of O1 / Manisa / Turkey / 69, A / Pocheon / 001 / KOR / 2010 and ASIA / Shamir / 89, : SEQ ID NOS: 1-6) was synthesized in a pHis-parallel vector and purified by using an Escherichia coli expression system. When the purified Multi-VP1e antigen protein was administered to mice, antibody formation to each serotype was confirmed by ELISA And SP ELISA confirmed the involvement of activation of T-immunoreactivity through ELIPOT, and demonstrated a high survival rate and low virus proliferation after foot-and-mouth disease infestation to show a successful vaccine effect -11).

In addition, the present inventors synthesized an expression vector, O / Jincheon, A / Pocheon, ASIA1 / Sharmir (Fig. 21: SEQ ID NO: 9-14), which is a currently prevalent serotype in addition to the three serotypes used in the above- One Multi-VP1e antigen protein was inoculated into mice by the same method. As a result, it was confirmed that the Multi-VP1e antigen protein composed of different serotypes was also effective as a vaccine against foot and mouth virus infection through antibody formation and T immune cell activation in serum (Fig. 21-28).

In addition, the present inventors have synthesized a VP1 multiepitope for a specific type of virus into an expression vector to express the Multi-VP1e antigen protein, confirming that the present invention can be used as a vaccine having an intensive effect on a specific type of virus. 29, SEQ ID NO: 17-22), foot-and-mouth disease virus type A / Pocheon, A / Malay97, A / Iraq (FIG. 35: SEQ ID NO: 25), O / Jincheon, O / Andong, O / Manisa (Fig. 41: SEQ ID NO: 33-38) were used for the synthesis of each Multi-VP1e antigen protein, and all types of Multi-VP1e In the experiment using the antigen protein, antibody formation in mouse serum and activation of T-immunoreactive cells were confirmed (Figs. 29-46).

In order to confirm the efficacy of the Multi-VP1e antigen protein in swine, a target animal of foot-and-mouth disease virus, the present inventors administered multi-VP1e antigen protein to pigs selected by SP ELISA on a farm, VP1e antigen protein was administered to the pigs. Multi-VP1e antigen protein was administered to pigs selected by the same method, and foot-and-mouth disease virus was infected to evaluate the clinical symptoms of foot-and-mouth disease virus The vaccine effect by the antigen protein was demonstrated (Fig. 12-20).

Accordingly, the Multi-VP1e antigen protein of the present invention can be useful for animals susceptible to foot-and-mouth disease virus as a vaccine against infectious disease of various types of serotypes.

Hereinafter, the present invention will be described in detail with reference to examples. It should be noted, however, that the following examples are provided to further illustrate the present invention and are not intended to limit the scope of the present invention.

Example  1. O1 manisa , A pocheon , Asia1 shamir  Recombinant linkage of major antigenic sites of each serotype Multi - VP1e  Gene production

Among the structural proteins of FMDV, the VP1 protein, which is known to be the most immunogenic, was selected as the target antigen protein. And further subdivided to produce one antigen capable of simultaneously enhancing immunogenicity for each serotype of O1 / Manisa / Turkey / 69, A / Pocheon / 001 / KOR / 2010 and Asia1 / Shamir / 89. Among the VP1 proteins, the GH loop (VP1 amino acid 132-162), which is known to have the greatest contribution to immunogenicity and has a very well conserved RGD motif between serotypes, and the C-terminus VP1 amino acid 192-212) (Fig. 1).

The sequence of each region was ligated using a linker amino acid sequence (GPGPG) to generate a gene sequence. The gene sequence was then synthesized to obtain the Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) Respectively.

Example  2. His tag - Multi - VP1e (O / Manisa -A / Pocheon - Asia / Shamir ) Preparation of Escherichia coli Expression Vector

The synthesized Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) gene was transfected with EcoRI at the 5 'end and XhoI site at the 3' end using the polymerase chain reaction (PCR). T Easy Vector (Invitrogen, Korea), followed by digestion with restriction enzymes inserted at 5 'and 3', and ligated again to pHis parallel vector. The His tag-Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) plasmid was transformed and amplified in E. coli DH5a and the amplified plasmid protein expression E. coli (E. coli BL21-CodonPlus DE3) -RIPL Competent Cells (Agilent technology, # 230280)) to induce the expression of the target protein (FIG. 2).

Example  3. E. coli  BL21- Codon Plus (DE3) - RIPL Competent Cell  of mine Target protein  Expression and purification

E. coli BL21-CodonPlus (DE3) -RIPL Competent Cells (Agilent technology, # 230280) transformed with His tag-Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) plasmid was transformed into LB Plates were streaked with platinum and incubated at 37 ° C for one day to induce colony formation. The colonies were seeded in 5 ml of LB broth, pre-incubated at 37 ° C for one day, and transferred to 3 L of LB broth. When the OD ₆₀₀ value reached about 0.6, the final concentration of IPTG was 1 mM. Protein formation was induced by stimulating the lactose-functioning gene using IPTG and cultured at 37 ° C for 4 hours. The cells were washed with buffer solution (50 mM Tris-HCl, 1 mM EDTA, pH 8.0), resuspended in the same buffer solution, and then sonicated to obtain the target protein (12,000 rpm / 30 min / 4 ° C), and only the supernatant was obtained to obtain a water-soluble protein. This water-soluble protein fraction was confirmed by Coomassie brilliant blue staining. Only the target protein antigens were purified using FPLC and IMAC using the identified water-soluble His tag-Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein.

Example  4. Refined His tag - Multi - VP1e (O / Manisa -A / Pocheon - Asia / Shamir ) Identification of protein

Purified target protein, Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein was electrophoresed on 12% SDS-PAGE and confirmed by Kumasi Brilliant Blue staining method. It was confirmed by Western blotting. The antibody used was an antibody made by using a peptide containing the RGD motif in VP1 of each serotype of O1 manisa, A pocheon, and Asia1 shamir (FIG. 3). These antibodies were reacted with PBST at a ratio of 1: 1000 to Western blotting, respectively, and the antigenic protein was identified at about 25 Kda, which is the combined size of His tag (3Kda) and Multi-VP1e (20Kda) 4).

Example  5. Antigen protein and  Manufacture of vaccine by mixing immunostimulant

It was confirmed that ISA201 obtained from SEPPIC led to the best immunity enhancement effect through previous experiments and this experiment was conducted using this. The antigen protein, Multi-VP1e, and immunostimulant were mixed at a weight ratio of 1: 1, and a 1-ml syringe and a 3-way stopcock were used. After mixing for 1 hour at room temperature, the mixture was stored at 4 ° C for one day.

Example  6. On the mouse FMDV  Immune response induction assay after vaccination

Five-week-old female C57BL / 6N crljori mice were purchased from Oriental Bio (Korea). The inoculation schedule of the multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein mixed with the immunostimulating agent proceeded as shown in FIG. 5, and the mice were divided into groups as shown in FIG. Experimental groups were divided into three groups: (1) ISA 201 and PBS buffer, (2) foot-and-mouth multi-VP1e and ISA 201, and (3) , And the efficacy of the vaccine multivalent antigen protein vaccine was tested.

In each group, 13 mice were isolated. In group 1), 40ul of PBS and 40ul of ISA201, 2) of group 2, 40ug of Multi-VP1e and 40ul of ISA201 were mixed and injected intramuscularly twice at intervals of one week. Group 3) was injected with 100ul of commercialized vaccine once a day in accordance with the method used by the Ministry of Agriculture, Forestry and Livestock Quarantine Division, Foot and Mouth Disease Diagnosis Division. The ELISA was performed by coating with an antigen peptide for O1 manisa, A pocheon, Asia1 shamir or Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) The generation rate was analyzed (Fig. 7). For more accurate analysis, the PrioCHECK® FMDV Type SP Antibody ELISA Kit (Life Technology), which is coated with inactivated FMDV, was performed for each serotype (FIG. 8).

As a result of the analysis at OD 450nm and the PrioCHECK® FMDV Type Antibody ELISA analysis, it was confirmed that Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) and ISA 201 mixture induce antibody production similar or higher than the commercial vaccine .

Example  7. FMDV  Analysis of vaccine efficacy against infection

The mice were finally infected with Multi-VP1e antigen protein by intraperitoneal administration of FMDV Asia 1 shamir virus 10LD ₅₀ in the mice two weeks after the inoculation. The serotype of Asia1 / shamir / 89 was used for the virus infection experiment in the mouse virus infection experiment, and it was conducted in the screening room for the foot and mouth disease diagnosis of the farm animal husbandry quarantine staff. The amount of virus was used as 10LC _50.

After 8 days of infection, the control mice died within 3 days, and all of the multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) and ISA 201 mixture and the commercial vaccine group survived 9).

On the 1st and 3rd day after FMDV Asia1 shamir virus infection, qRT-PCR was performed on the mouse serum samples. As a result, viruses were completely eliminated from the third day in the vaccination group, while the multi-VP1e multivalent antigen protein- It was confirmed that complete removal occurred from the beginning (Fig. 10). Thus, it was confirmed that the vaccine efficacy of Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein was excellent.

Example  8. Mouse In splenocytes  Cytokine production analysis

In order to investigate whether T-immunoreactive cells were involved in the immune response by multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein, spleen splenocytes from mice inoculated with Multi-VP1e antigen protein were isolated VP1 peptides such as O1 / Manisa / Turkey / 69, A / Pocheon / 001 / KOR / 2010 and Asia1 / Shamir / 89 T-immune cell response experiments were performed using stimulation.

Experiments were conducted using a BD ^TM ELISPOT set. First, purified IFN-r antibody and purified IL-4 antibody were diluted 1: 200 in PBS and dispensed in 100 ul / well. The next day, splenocytes were isolated and spleen cells were obtained. Then, red blood cells were removed using ACK lysis buffer, followed by washing with RPMI media and resuspension. The number of cells in the media was counted, and the splenocytes obtained from each individual were equally adjusted to a concentration of 7 × ¹⁰ 5/100 μl and then dispensed at 100 μl / well. The stimulants (VP1 peptides) were equally diluted in RPMI medium (10% FBS) at a concentration of 3 ug / 100 ul and dispensed at 100 ul / well. After incubation for 24 hours at 37 ° C in a 5% CO ₂ incubator, the detection antibody and Streptabidin-HRP were reacted. Finally, it was developed using AEC buffer, and the number of points generated by IFN-r and IL-4 was measured and expressed in a graph (FIG. 11). It was confirmed that the experiment was correctly conducted through the positive control group, Mitogen stimulant (1 ug / well) and the negative control media only, and it was confirmed that IFN-r and IL-4 were produced only in the vaccine group, . These results demonstrate that the multi-VP1e antigen protein contributes to the activation of T-immune cells against the multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein.

Example  9. On the pig Multi - VP1e Antigen protein  Immunogenicity analysis after inoculation

Twenty ewes of 8-week-old pigs raised on the farm were screened using the PrioCHECK® FMDV Type O SP Atibody ELISA Kit (FIG. 12). In the screening test, 12 pigs were excluded from the pigs with high percentage inhibition (PI) values and divided into 4 groups of 3 pigs. A control group was set up by administering a mixture of immunopotentiator and PBS buffer. Three dose groups were set up with 100 μg, 200 μg and 300 μg of Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) (Fig. 14).

In accordance with the administration schedule of FIG. 13, the multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein was administered at 0 and 4 weeks and the blood was collected at 3 and 6 weeks. Pig blood was stored at 4 ° C for 1 hour, and serum was obtained using a centrifuge. Antigen-coated ELISA was performed using porcine serum at 6 weeks. Serum was diluted sequentially from 1:50 to 2: 1, and dilutions were set up to 1: 1600.

The vaccine group showed high antibody values for all antigens such as Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein and VP1 peptide of O1 manisa, A pocheon and Asia1 shamir virus. (Fig. 15). Porcine sera were performed for each serotype of O, A, and Asia using the PrioCHECK® FMDV Type SP Antibody ELISA. The ELISA after the first and second inoculation showed high values of the multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein inoculation group and individual differences were also reduced (Fig. 16) .

Example  10. In pigs Multi - VP1e  Antigen protein of  Vaccine efficacy analysis

Fifteen 8-week-old pigs fed on the farm were screened using the PrioCHECK® FMDV Type O SP Antibody ELISA Kit. (Fig. 17). As a result of the screening test, eleven pigs were excluded from pigs having a high PI value, and an experimental plan was established as shown in FIG. Inoculation of the commercial vaccine and Multi-VP1e (O / Manisa-A / Pocheon-sia / Shamir) antigen protein was performed at 0 and 4 weeks after infection.

As shown in FIG. 19, no change in the body temperature of the commercial vaccination group and the multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein inoculation group was observed. As a result of confirming clinical symptoms such as blister formation, appetite loss, leg dissection, and inability to stand on mouth or foot for 10 days after the inoculation of Asia 1 type Shamir strain, the clinical symptoms shown in the Adjuvant only group were commercialized Vaccine group and Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein inoculation group.

The vaccine candidate prepared by mixing 300 ug of Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) antigen protein with ISA201 VG according to the results of this experiment was shown to protect against FMDV attack in pigs as well as mouse models And that the immune system can induce an immune response.

Example  11- 14. Various foot-and-mouth disease viruses Protection effect against subtype

Example  11. New foot-and-mouth disease On a weekly basis  Confirm efficacy for

11-1. New foot-and-mouth disease incidence Multi - VP1e  Antigen protein production

A new Multi-VP1e can be constructed as an epitope in VP1 of foot-and-mouth disease virus, which is currently being developed, to verify vaccine efficacy and to produce a vaccine that can be used to prevent foot-and-mouth disease. (O / Jincheon), A / Pocheon, and Asia1 / Shamir GH loop (VP1) of three serotypes by replacing the O1 manisa of the serotype of Example 1 with the O-type serotype O / amino acid 132 to 162) and C-terminus (VP1 amino acid 192 to 212) (FIG. 21) ) Gene.

11-2. His tag - Multi - VP1e (O / Jincheon -A / Pocheon - Asia / Shamir ) Production of Escherichia coli Expression Vector, Target protein  Expression and purification

(SEQ ID NO: 1) -RIPL (DE3) -RIPL (DE3) -RIPL (DE3) -RIPL (SEQ ID NO: 2) was prepared by the same method as in Example 2 except that the EcoRI site was inserted at the 5 'end and the XhoI site was inserted at the 3' Competent Cells (Agilent technology, # 230280) (Agilent technology) to induce expression of the target protein. A soluble target protein was isolated from E. coli through the method of Example 3 and the degree of expression was confirmed. The identified water-soluble His tag-Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) antigen protein was purified using FPLC and IMAC.

11-3. Refined His tag - Multi - VP1e (O / Jincheon -A / Pocheon - Asia / Shamir ) Identification of protein

The purified target protein Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) antigen protein was identified by the Kumishi Brilliant Blue staining method as in Example 4. The A VP1 used in Example 4, the RGD The expression of the antigen protein was confirmed by Western blot using the motif antibody (Fig. 23).

11-4. Identification of immunogenicity against FMD in mice

The multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) protein and the immunostimulant were mixed at a weight ratio of 1: 1 as in Example 5, the mouse group was divided as shown in FIG. 24, Respectively. Serum was obtained from each mouse on the 21st day after the start of inoculation as in Example 6 and coated with antigen peptide or multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) protein for O jincheon, A pocheon and Asia 1 shamir The antibody production rate was analyzed at OD450nm through an ELISA (Fig. 25). The Type SP Antibody ELISA Kit (Life Technology) was also performed for each serotype (FIG. 26). As a result of the two antibody production analysis, it was confirmed that Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) and ISA 201 mixture induce high antibody production similar to the commercial vaccine.

11-5. Antigen protein of  Vaccine efficacy analysis

FMDV virus by administering Asia1 shamir 10LD ₅₀ both after two weeks in a mouse inoculated with the Multi-VP1e (O / Jincheon- A / Pocheon-Asia / Shamir) antigen proteins last day into the abdominal cavity of a mouse to induce infection. The survival rate of 8 days after infection, the control mice died on the 2nd day, and only one dog died from Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) All survived (Fig. 27). It was confirmed that the vaccine efficacy of Multi-VP1e antigen protein was excellent.

11-6. mouse In splenocytes  Cytokine production assay

(O / Jincheon-A / Pocheon) as in Example 8 to examine whether T-immunoreactive cells were involved in the immune response by the multi-VP1e (O / Jincheon-A / Pocheon- Splenic splenocytes from mice inoculated with Asia / Shamir antigen protein were isolated and cultured in the presence of Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) protein, O / Jincheon, A / Pocheon / 001 / KOR / 2010 and Asia1 / Shamir / 89 were used for ELISPOT. As a result of measuring the degree of secretion of IFN-r and IL-4, IFN-r and IL-4 were produced only in the vaccine group (FIG. 28). In this study, we investigated the effect of multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) and O jincheon, A pocheon, Asia 1 shamir antigen And to the activated immune response of T - immune cells to the protein. Through this experiment, we confirmed that it is possible to produce a vaccine that can be used for preventing foot-and-mouth disease by applying a new Multi-VP1e to the foot-and-mouth disease virus.

Example  12. Foot and mouth disease virus O type  Check the efficacy of Korean vaccine

12-1. Foot-and-mouth disease virus O type Multi - VP1e  Antigen protein production

By constructing a new Multi-VP1e with the VP1 epitope of a specific type FMDV, the vaccine can be verified and applied to produce a vaccine that can be used to prevent foot-and-mouth disease. (VP1 amino acid 132-162) and the C-terminus (VP1 amino acid 192-212) of the O-type FM virus O (J / O / Jincheon, O / Andong, 29), and the gene was named Multi-VP1e (O / Jincheon, O / Andong, O / Manisa).

12-2. His tag - Multi - VP1e  (O / Jincheon , O / Andong , O / Manisa ) Production of Escherichia coli Expression Vector, Target protein  Expression, purification and identification

JC-Multi-VP1 plasmid was constructed by PCR (EcoRI) at the 5 'end and XhoI site at the 3' end and PCR was performed. E. coli BL21-CodonPlus (DE3) -RIPL Competent Cells , # 230280) to induce expression of the target protein. A soluble target protein was isolated from E. coli through the method of Example 3 and the degree of expression was confirmed. Identified soluble His tag-Multi-VP1e (O / Jincheon, O / Andong, O / Manisa) antigen protein was purified using FPLC and IMAC. The purified target protein Multi-VP1e (O / Jincheon, O / Andong, O / Manisa) antigen protein was identified by Coomassie brilliant blue staining method as in Example 4 and O / Jincheon, O / Andong, O / Manisa VP1 The expression of the antigen protein was confirmed by Western blot using the inner RGD motif antibody (Fig. 31) (Fig. 32).

12-3. Immunogenicity analysis for mouse FMD in mice

As in Example 5, Multi-VP1e (O / Jincheon, O / Andong, O / Manisa) protein and immunostimulant were mixed at a weight ratio of 1: Serum was obtained from each mouse on the 21st day after the start of inoculation as in Example 6, and antigenic peptides or O / Jincheon, O / Andong, O / Manisa proteins for O / Jincheon, O / The antibody production rate was analyzed at OD450nm by ELISA performed by coating (Fig. 33). As a result of the antibody production analysis, it was confirmed that the multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) and ISA 201 mixture produced antibody similar to the commercial vaccine. In addition, the reaction of T-immunoreactive cells was analyzed in the same manner as in Example 8, confirming that the immune response of specific T-immunoreactive cells was induced by Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) 34).

Example  13. Foot-and-mouth disease virus A type  Check the efficacy of Korean vaccine

13-1. Foot-and-mouth disease virus A type Multi - VP1e  Antigen protein production

(VP1 amino acid 132-162) and the C-terminus (VP1 amino acid 192-212) of three types of foot-and-mouth disease virus type O (A), and A / Pocheon 35), and the gene was prepared. We named this gene as Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq).

13-2. His tag - Multi - VP1e  (A / Pocheon , A / Malay97 , A / Iraq ) Production of Escherichia coli Expression Vector, Target protein  Expression, purification and identification

JC-Multi-VP1 plasmid was constructed by PCR (EcoRI) at the 5 'end and XhoI site at the 3' end and PCR was performed. E. coli BL21-CodonPlus (DE3) -RIPL Competent Cells , # 230280) to induce expression of the target protein. A soluble target protein was isolated from E. coli through the method of Example 3 and the degree of expression was confirmed. Identified soluble His tag-Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) antigen protein was purified using FPLC and IMAC. Purified target protein Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) antigen protein was identified by Coomassie brilliant blue staining method as in Example 4, and A / Pocheon, A / Malay97, Using the RGD motif antibody (FIG. 37), the expression of the antigen protein was confirmed by Western blot (FIG. 38).

13-3. Immunogenicity analysis for mouse FMD in mice

As in Example 5, Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) protein and immunostimulant were mixed at a weight ratio of 1: Serum was obtained from each mouse on the 21st day after the start of inoculation as in Example 6, and the antigen peptide or Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) protein for A / Pocheon, A / Malay97 and A / The antibody production rate was analyzed at OD450nm by ELISA performed by coating (Fig. 39). As a result of the antibody production analysis, it was confirmed that Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) and ISA201 mixture induce high antibody production similar to the commercial vaccine. In addition, the reaction of T-immunized cells was analyzed by the same method as in Example 8 to confirm that an immune response of specific T-immunized cells was induced by Multi-VP1e (A / Pocheon, A / Malay97, A / )

Example  14. Foot and mouth virus Asia type  Check the efficacy of Korean vaccine

14- 1. foot and mouth disease  virus Asia type Multi - VP1e Antigen protein  making

The antigen sites including the GH loop (VP1 amino acid 132-162) and the C-terminus (VP1 amino acid 192-212) of three types of foot-and-mouth disease virus type Asia were determined 41), and this gene was named Multi-VP1e (Asia / Shamir, Asia / Mongolia05, Asia / LC04) gene.

14-2. His tag - Multi - VP1e  ( Asia / Shamir , Asia / Mongolia05 , Asia / LC04) Construction of Escherichia coli Expression Vector, Target protein  Expression, purification and identification

JC-Multi-VP1 plasmid was constructed by PCR (EcoRI) at the 5 'end and XhoI site at the 3' end and PCR was performed. E. coli BL21-CodonPlus (DE3) -RIPL Competent Cells , # 230280) to induce expression of the target protein. A soluble target protein was isolated from E. coli through the method of Example 3 and the degree of expression was confirmed. Identified soluble His tag-Multi-VP1e (Asia / Shamir, Asia / Mongolia05, Asia / LC04) antigen protein was purified using FPLC and IMAC. Purified target protein Multi-VP1e (Asia / Shamir, Asia / Mongolia05, Asia / LC04) antigen protein was identified by Kumasi Brilliant Blue staining method as in Example 4, and Asia / Shamir, Asia / Mongolia 05, Asia / LC04 VP1 The expression of the antigen protein was confirmed by Western blot using the inner RGD motif antibody (Fig. 43) (Fig. 44).

14- 3. Mouse  Immunogenicity analysis of mycoplasma antigen

As in Example 5, Multi-VP1e (Asia / Shamir, Asia / Mongolia 05, Asia / LC04) protein and immunostimulant were mixed at a weight ratio of 1: Serum was obtained from each mouse on the 21st day after the start of inoculation as in Example 6, and the antigen peptide or Multi-VP1e (Asia / Shamir, Asia / Mongolia 05, Asia / LC04) protein for Asia / Shamir, Asia / Mongolia 05, Asia / LC04 The antibody production rate was analyzed at OD450nm by ELISA performed by coating (FIG. 45). As a result of the antibody production analysis, it was confirmed that Multi-VP1e (Asia / Shamir, Asia / Mongolia05, Asia / LC04) and ISA 201 mixture induce high antibody production similar to the commercial vaccine. In addition, it was confirmed that the immune response of specific T-immunoreactive cells was induced by Multi-VP1e (Asia / Shamir, Asia / Mongolia 05, Asia / LC04) by analyzing the reaction of T immune cells in the same manner as in Example 8 46).

<110> Animal and Plant Quaratine Agency And Chungnam National University <120> Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and          Uses Thereof <130> 16-10602 <160> 43 <170> KoPatentin 3.0 <210> 1 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 1 Gly Asn Cys Lys Tyr Gly Asp Gly Thr Val Ala Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Ala Leu Pro Thr Ser              20 25 30 <210> 2 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 2 Leu Ala Ile His Pro Asp Gln Ala Arg His Lys Gln Lys Ile Val Ala   1 5 10 15 Pro Val Glu Gln Leu              20 <210> 3 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 3 Val Tyr Asn Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly   1 5 10 15 Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala              20 25 30 <210> 4 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 4 Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys Ile   1 5 10 15 Ile Ala Pro Ala Lys              20 <210> 5 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 5 Tyr Asn Gly Lys Thr Ala Tyr Gly Glu Thr Thr Ser Arg Arg Gly Asp   1 5 10 15 Met Ala Ala Leu Ala Gln Arg Leu Ser Ala Arg Leu Pro Thr Ser              20 25 30 <210> 6 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 6 Leu Ala Leu Asp Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala   1 5 10 15 Pro Gln Lys Gln Val              20 <210> 7 <211> 543 <212> DNA <213> Artificial Sequence <220> Polynucleotide of Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) <400> 7 ggcaactgca aatatggcga tggcaccgtg gcgaacgtgc gtggcgatct gcaggtgctg 60 gcgcagaaag cggcgcgtgc gctgccgacc agcggcccgg gcccgggcct ggcgattcat 120 ccggatcagg cgcgtcataa acagaaaatt gtggcgccgg tggaacagct gggcccgggc 180 ccgggcgtgt ataacggcac cagccgttat agcgcgccgg cgacccgtcg tggcgatctg 240 ggcagcctgg cggcgcgtct ggcggcgcag ctgccggcgg gcccgggccc gggcctgctg 300 gcggtggaag tgaccagcca ggatcgtcat aaacagaaaa ttattgcgcc ggcgaaaggc 360 ccgggcccgg gctataacgg caaaaccgcg tatggcgaaa ccaccagccg tcgtggcgat 420 atggcggcgc tggcgcagcg tctgagcgcg cgtctgccga ccagcggccc gggcccgggc 480 ctggcgctgg ataccaccca ggatcgtcgt aaacaggaaa ttattgcgcc gcagaaacag 540 gtg 543 <210> 8 <211> 181 <212> PRT <213> Artificial Sequence <220> Synthetic protein Multi-VP1e (O / Manisa-A / Pocheon-Asia / Shamir) <400> 8 Gly Asn Cys Lys Tyr Gly Asp Gly Thr Val Ala Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Ala Leu Pro Thr Ser Gly              20 25 30 Pro Gly Pro Gly Leu Ala Ile His Pro Asp Gln Ala Arg His Lys Gln          35 40 45 Lys Ile Val Ala Pro Val Glu Gln Leu Gly Pro Gly Pro Gly Val Tyr      50 55 60 Asn Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly Asp Leu  65 70 75 80 Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Gly Pro Gly                  85 90 95 Pro Gly Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His Lys Gln             100 105 110 Lys Ile Ale Pro Ala Lys Gly Pro Gly Pro Gly Tyr Asn Gly Lys         115 120 125 Thr Ala Tyr Gly Glu Thr Thr Ser Arg Gly Asp Met Ala Ala Leu     130 135 140 Ala Gln Arg Leu Ser Ala Arg Leu Pro Thr Ser Gly Pro Gly Pro Gly 145 150 155 160 Leu Ala Leu Asp Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala                 165 170 175 Pro Gln Lys Gln Val             180 <210> 9 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 9 Gly Asn Cys Lys Tyr Thr Gly Gly Ser Leu Pro Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro Thr Ser              20 25 30 <210> 10 <211> 20 <212> PRT <213> Foot-and-mouth disease virus <400> 10 Leu Ala Val His Ser Ser Ala Ala Arg His Lys Gln Lys Ile Val Ala   1 5 10 15 Pro Val Lys Gln              20 <210> 11 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 11 Val Tyr Asn Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly   1 5 10 15 Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala              20 25 30 <210> 12 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 12 Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys Ile   1 5 10 15 Ile Ala Pro Ala Lys              20 <210> 13 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 13 Val Tyr Asn Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly   1 5 10 15 Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala              20 25 30 <210> 14 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 14 Leu Ala Leu Asp Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala   1 5 10 15 Pro Gln Lys Gln Val              20 <210> 15 <211> 540 <212> DNA <213> Artificial Sequence <220> Polynucleotide of Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) <400> 15 ggtaattgta aatatactgg tggttcttta cctaatgttc gtggtgattt acaagtttta 60 gctcctaaag ctgctcgtcc tttacctact tctggtcctg gtcctggttt agctgttcat 120 ccttctgctg ctcgtcataa acaaaaaatt gttgctcctg ttaaacaagg tcctggtcct 180 ggtgtttata atggtacttc tcgttattct gctcctgcta ctcgtcgtgg tgatttaggt 240 tctttagctg ctcgtttagc tgctcaatta cctgctggtc ctggtcctgg tttattagct 300 gttgaagtta cttctcaaga tcgtcataaa caaaaaatta ttgctcctgc taaaggtcct 360 ggtcctggtt ataatggtaa aactgcttat ggtgaaacta cttctcgtcg tggtgatatg 420 gctgctttag ctcaacgttt atctgctcgt ttacctactt ctggtcctgg tcctggttta 480 gctttagata ctactcaaga tcgtcgtaaa caagaaatta ttgctcctca aaaacaagtt 540                                                                          540 <210> 16 <211> 180 <212> PRT <213> Artificial Sequence <220> Synthetic protein Multi-VP1e (O / Jincheon-A / Pocheon-Asia / Shamir) <400> 16 Gly Asn Cys Lys Tyr Thr Gly Gly Ser Leu Pro Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro Thr Ser Gly              20 25 30 Pro Gly Pro Gly Leu Ala Val His Pro Ser Ala Ala Arg His Lys Gln          35 40 45 Lys Ile Val Ala Pro Val Lys Gln Gly Pro Gly Pro Gly Val Tyr Asn      50 55 60 Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly Asp Leu Gly  65 70 75 80 Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Gly Pro Gly Pro                  85 90 95 Gly Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys             100 105 110 Ile Ile Pro Ala Lys Gly Pro Gly Pro Gly Tyr Asn Gly Lys Thr         115 120 125 Ala Tyr Gly Glu Thr Thr Ser Arg Gly Asp Met Ala Ala Leu Ala     130 135 140 Gln Arg Leu Ser Ala Arg Leu Pro Thr Ser Gly Pro Gly Pro Gly Leu 145 150 155 160 Ala Leu Asp Thr Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala Pro                 165 170 175 Gln Lys Gln Val             180 <210> 17 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 17 Gly Asn Cys Lys Tyr Thr Gly Gly Ser Leu Pro Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro Thr Ser              20 25 30 <210> 18 <211> 20 <212> PRT <213> Foot-and-mouth disease virus <400> 18 Leu Ala Val His Ser Ser Ala Ala Arg His Lys Gln Lys Ile Val Ala   1 5 10 15 Pro Val Lys Gln              20 <210> 19 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 19 Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro Thr Ser              20 25 30 <210> 20 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 20 Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys Ile   1 5 10 15 Ile Ala Pro Ala Lys              20 <210> 21 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 21 Gly Asn Cys Lys Tyr Gly Asp Gly Thr Val Ala Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Ala Leu Pro Thr Ser              20 25 30 <210> 22 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 22 Leu Ala Ile His Pro Asp Gln Ala Arg His Lys Gln Lys Ile Val Ala   1 5 10 15 Pro Val Glu Gln Leu              20 <210> 23 <211> 540 <212> DNA <213> Artificial Sequence <220> Polynucleotide of Multi-VP1e (O / Jincheon, O / Andong, O / Manisa) <400> 23 ggcaactgca aatataccgg cggcagctta ccgaacgtgc gcggcgattt acaggtgtta 60 gcgccgaaag cggcgcgccc gttaccgacc agcggtcctg gtcctggttt agcggtgcat 120 ccgagcgcgg cgcgccataa acagaaaatt gtggcgccgg tgaaacaggg tcctggtcct 180 ggtggcaact gcaaatatgc gggcggcagc ttaccgaacg tgcgcggcga tttacaggtg 240 ttagcgcaga aagcggcgcg cccgttaccg accagcggtc ctggtcctgg tttagcggtg 300 catccgagcg cggcgcgcca taaacagaaa attgtggcgc cggtgaaaca gagcggtcct 360 ggtcctggtg gcaactgcaa atatggcgat ggcaccgtgg cgaacgtgcg cggcgattta 420 caggtgttag cgcagaaagc ggcgcgcgcg ttaccgacca gcggtcctgg tcctggttta 480 gcgattcatc cggatcaggc gcgccataaa cagaaaattg tggcgccggt ggaacagtta 540                                                                          540 <210> 24 <211> 180 <212> PRT <213> Artificial Sequence <220> Synthetic protein Multi-VP1e (O / Jincheon, O / Andong, O / Manisa) <400> 24 Gly Asn Cys Lys Tyr Thr Gly Gly Ser Leu Pro Asn Val Arg Gly Asp   1 5 10 15 Leu Gln Val Leu Ala Pro Lys Ala Ala Arg Pro Leu Pro Thr Ser Gly              20 25 30 Pro Gly Pro Gly Leu Ala Val His Pro Ser Ala Ala Arg His Lys Gln          35 40 45 Lys Ile Val Ala Pro Val Lys Gln Gly Pro Gly Pro Gly Gly Asn Cys      50 55 60 Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val Arg Gly Asp Leu Gln Val  65 70 75 80 Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro Thr Ser Gly Pro Gly Pro                  85 90 95 Gly Leu Ala Val His Ser Ser Ala Ala Arg His Lys Gln Lys Ile Val             100 105 110 Ala Pro Val Lys Gln Ser Gly Pro Gly Pro Gly Gly Asn Cys Lys Tyr         115 120 125 Gly Asp Gly Thr Val Ala Asn Val Arg Gly Asp Leu Gln Val Leu Ala     130 135 140 Gln Lys Ala Ala Arg Ala Leu Pro Thr Ser Gly Pro Gly Pro Gly Leu 145 150 155 160 Ala Ile His Pro Asp Gln Ala Arg His Lys Gln Lys Ile Val Ala Pro                 165 170 175 Val Glu Gln Leu             180 <210> 25 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 25 Val Tyr Asn Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly   1 5 10 15 Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala              20 25 30 <210> 26 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 26 Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His Lys Gln Lys Ile   1 5 10 15 Ile Ala Pro Ala Lys              20 <210> 27 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 27 Gly Thr Ser Lys Tyr Ser Thr Pro Gly Ala Arg Arg Gly Asp Leu Gly   1 5 10 15 Ser Leu Ala Ala Arg Asp Ala Ala Gln Leu Pro Ala Ser Phe Asn              20 25 30 <210> 28 <211> 19 <212> PRT <213> Foot-and-mouth disease virus <400> 28 Val Glu Val Leu Ser Gln Asp Arg His Lys Gln Arg Ile Ile Ala Pro   1 5 10 15 Ala Lys Gln             <210> 29 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 29 Gly Thr Ser Lys Tyr Ser Ala Gly Gly Thr Gly Arg Arg Gly Asp Leu   1 5 10 15 Gly Pro Leu Ala Ala Arg Val Ala Ala Gln Leu Pro Ala Ser Phe              20 25 30 <210> 30 <211> 20 <212> PRT <213> Foot-and-mouth disease virus <400> 30 Ala Val Glu Val Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala   1 5 10 15 Pro Ala Lys Gln              20 <210> 31 <211> 534 <212> DNA <213> Artificial Sequence <220> Polynucleotide of Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) <400> 31 gtgtataacg gcaccagccg ttatagcgcg ccggcgaccc gtcgtggcga tctgggcagc 60 ctggcggcgc gtctggcggc gcagctgccg gcgggcccgg gcccgggcct gctggcggtg 120 gaagtgacca gccaggatcg tcataaacag aaaattattg cgccggcgaa aggcccgggc 180 ccgggcggca ccagcaaata tagcaccccg ggcgcgcgtc gtggcgatct gggcagcctg 240 gcggcgcgtg atgcggcgca gctgccggcg agctttaacg gcccgggccc gggcgtggaa 300 gtgctgagcc aggatcgtca taaacagcgt attattgcgc cggcgaaaca gggcccgggc 360 ccgggcggca ccagcaaata tagcgcgggc ggcaccggcc gtcgtggcga tctgggcccg 420 ctggcggcgc gtgtggcggc gcagctgccg gcgagctttg gcccgggccc gggcgcggtg 480 gaagtgagca gccaggatcg tcataaacag aaaattattg cgccggcgaa acag 534 <210> 32 <211> 178 <212> PRT <213> Artificial Sequence <220> Synthetic protein Multi-VP1e (A / Pocheon, A / Malay97, A / Iraq) <400> 32 Val Tyr Asn Gly Thr Ser Arg Tyr Ser Ala Pro Ala Thr Arg Arg Gly   1 5 10 15 Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Gly              20 25 30 Pro Gly Pro Gly Leu Leu Ala Val Glu Val Thr Ser Gln Asp Arg His          35 40 45 Lys Gln Lys Ile Ale Pro Ala Lys Gly Pro Gly Pro Gly Gly Thr      50 55 60 Ser Lys Tyr Ser Thr Pro Gly Ala Arg Arg Gly Asp Leu Gly Ser Leu  65 70 75 80 Ala Ala Arg Asp Ala Ala Gln Leu Pro Ala Ser Phe Asn Gly Pro Gly                  85 90 95 Pro Gly Val Glu Val Leu Ser Gln Asp Arg His Lys Gln Arg Ile Ile             100 105 110 Ala Pro Ala Lys Gln Gly Pro Gly Pro Gly Gly Thr Ser Lys Tyr Ser         115 120 125 Ala Gly Gly Thr Gly Arg Arg Gly Asp Leu Gly Pro Leu Ala Ala Arg     130 135 140 Val Ala Ala Gln Leu Pro Ala Ser Phe Gly Pro Gly Pro Gly Ala Val 145 150 155 160 Glu Val Ser Ser Gln Asp Arg His Lys Gln Lys Ile Ile Ala Pro Ala                 165 170 175 Lys Gln         <210> 33 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 33 Tyr Asn Gly Lys Thr Ala Tyr Gly Glu Thr Thr Ser Arg Arg Gly Asp   1 5 10 15 Met Ala Ala Leu Ala Gln Arg Leu Ser Ala Arg Leu Pro Thr Ser              20 25 30 <210> 34 <211> 21 <212> PRT <213> Foot-and-mouth disease virus <400> 34 Leu Ala Leu Asp Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala   1 5 10 15 Pro Gln Lys Gln Val              20 <210> 35 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 35 Lys Thr Thr Tyr Gly Glu Glu Ser Ser Arg Gly Asp Leu Ala Ala   1 5 10 15 Leu Ala Arg Arg Val Asn Asn Arg Leu Pro Thr Ser Phe Asn Tyr              20 25 30 <210> 36 <211> 17 <212> PRT <213> Foot-and-mouth disease virus <400> 36 Asp Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala Pro Glu Lys   1 5 10 15 Gln     <210> 37 <211> 31 <212> PRT <213> Foot-and-mouth disease virus <400> 37 Lys Thr Ala Tyr Gly Glu Thr Thr Thr Arg Arg Gly Asp Leu Ala Ala   1 5 10 15 Leu Ala Gln Arg Val Ser Ser Gln Leu Pro Thr Ser Phe Asn Tyr              20 25 30 <210> 38 <211> 17 <212> PRT <213> Foot-and-mouth disease virus <400> 38 Asp Thr Val Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala Pro Glu Lys   1 5 10 15 Gln     <210> 39 <211> 519 <212> DNA <213> Artificial Sequence <220> Polynucleotide of Multi-VP1e (Asia / Shamir, Asia / Mongolia 05,          Asia / LC04) <400> 39 tataacggca aaaccgcgta tggcgaaacc accagccgtc gtggcgatat ggcggcgctg 60 gcgcagcgtc tgagcgcgcg tctgccgacc agcggcccgg gcccgggcct ggcgctggat 120 accacccagg atcgtcgtaa acaggaaatt attgcgccgc agaaacaggt gggcccgggc 180 ccgggcaaaa ccacctatgg cgaagaaagc agccgtcgtg gcgatctggc ggcgctggcg 240 cgtcgtgtga acaaccgtct gccgaccagc tttaactatg gcccgggccc gggcgatacc 300 acccaggatc gtcgtaaaca ggaaattatt gcgccggaaa aacagggccc gggcccgggc 360 aaaaccgcgt atggcgaaac caccacccgt cgtggcgatc tggcggcgct ggcgcagcgt 420 gtgagccgtc agctgccgac cagctttaac tatggcccgg gcccgggcga taccgtgcag 480 gatcgtcgta aacaggaaat tattgcgccg gaaaaacag 519 <210> 40 <211> 173 <212> PRT <213> Artificial Sequence <220> Synthetic protein Multi-VP1e (Asia / Shamir, Asia / Mongolia 05,          Asia / LC04) <400> 40 Tyr Asn Gly Lys Thr Ala Tyr Gly Glu Thr Thr Ser Arg Arg Gly Asp   1 5 10 15 Met Ala Ala Leu Ala Gln Arg Leu Ser Ala Arg Leu Pro Thr Ser Gly              20 25 30 Pro Gly Pro Gly Leu Ala Leu Asp Thr Thr Gln Asp Arg Arg Lys Gln          35 40 45 Glu Ile Ile Ala Pro Gln Lys Gln Val Gly Pro Gly Pro Gly Lys Thr      50 55 60 Thr Tyr Gly Glu Glu Ser Ser Arg Gly Asp Leu Ala Ala Leu Ala  65 70 75 80 Arg Arg Val Asn Asn Arg Leu Pro Thr Ser Phe Asn Tyr Gly Pro Gly                  85 90 95 Pro Gly Asp Thr Thr Gln Asp Arg Arg Lys Gln Glu Ile Ile Ala Pro             100 105 110 Glu Lys Gln Gly Pro Gly Pro Gly Lys Thr Ala Tyr Gly Glu Thr Thr         115 120 125 Thr Arg Gly Asp Leu Ala Leu Ala Gln Arg Val Ser Ser Gln     130 135 140 Leu Pro Thr Ser Phe Asn Tyr Gly Pro Gly Pro Gly Asp Thr Val Gln 145 150 155 160 Asp Arg Arg Lys Gln Glu Ile Ile Ala Pro Glu Lys Gln                 165 170 <210> 41 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 41 Gly Pro Gly Pro Gly   1 5 <210> 42 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 42 Gly Gly Gly Gly Gly   1 5 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Peptide linker <400> 43 Gly Gly Gly Gly Pro   1 5

Claims (21)

A soluble Multi-Vp1e antigen protein comprising a plurality of epitopes according to the serotype of foot-and-mouth disease virus (FMDV) VP1 protein,
Wherein the soluble Multi-Vp1e antigen protein comprises an RGD motif and a C-terminus epitope according to three serotypes, wherein the soluble Multi-VP1e antigen protein comprises the sequence of SEQ ID NOs: 17 to 22, The soluble Multi-VP1e antigen protein of any one of claims 17 to 22 is linked via a peptide linker of the Gly-Pro-Gly-Pro-Gly sequence (SEQ ID NO: 41), respectively. delete delete delete delete delete 2. The soluble multi-VP1e antigen protein of claim 1, wherein the soluble Multi-VP1e antigen protein is capable of inducing a humoral immune response or a cellular immune response. 9. A polynucleotide encoding the soluble Multi-VP1e antigen protein of any one of claims 1 or 7. 9. The polynucleotide of claim 8, wherein the polynucleotide is modified to match the codon usage of E. coli. 10. The polynucleotide according to claim 9, which is a nucleotide sequence of SEQ ID NO: 23. 10. A recombinant vector comprising the polynucleotide of claim 10. 12. Recombinant microorganism transformed with the vector of claim 11. 13. The recombinant microorganism according to claim 12, wherein the microorganism is E. coli. 14. The recombinant microorganism of claim 13, wherein the E. coli is BL21-Codon Plus (DE3) -RIPL cells. (a) preparing a recombinant vector comprising the polynucleotide of claim 8;
(b) transforming and culturing Escherichia coli with the vector;
(c) disrupting E. coli cultured in step (b) to obtain a Multi-VP1e antigen protein. 16. The method of mass production according to claim 15, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 23. 16. The method of mass production according to claim 15, wherein the E. coli is BL21-Codon Plus (DE3) -RIPL cells. A vaccine composition for preventing and treating FMD virus comprising the soluble Multi-VP1e antigen protein of any one of claims 1 to 7 as an active ingredient. 19. The vaccine composition of claim 18, further comprising a pharmaceutically acceptable carrier or adjuvant. 9. A method for enhancing immunity against foot-and-mouth disease virus comprising administering to a subject a soluble Multi-VP1e antigen protein of any one of claims 1 or 7. delete

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