KR20180068936A - Soluble Multi-Epitope Antigen of Foot-and-Mouth Disease Virus and Uses Thereof - Google Patents

Abstract

The present invention relates to a vector which expresses fusion protein antigens (multi-VP1 epitopes) into soluble protein by using a specific microorganism expression system after performing codon optimization of main antigenic epitopes in VP1 protein of foot-and-mouth disease having different blood serum types and joining the main antigenic epitopes, a microorganism transformed by the vector, soluble multi-VP1 epitope protein expressed and refined from the microorganism, and use of the protein for the purpose of a vaccine. More specifically, the present invention relates to an Escherichia coli expression vector comprising a gene in which main antigenic epitopes in VP1 protein of O, A, and Asia1 types of foot-and-mouth disease virus with a gene sequence, changed to be optimized for codon of Escherichia coli, or different local types of foot and mouth disease in each same blood serum type are joined, a specific recombinant Escherichia coli transformed by the vector, and a vaccine for preventing infection from foot-and-mouth disease, the vaccine having soluble multi-VP1 epitope protein, expressed and refined from the Escherichia coli, 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 virus with different serotypes, and more particularly, to a fusion protein antigen (Multi-VP1 epitopes) using a specific E. coli expression system. It relates to a vaccine for preventing foot-and-mouth disease virus infection, containing a vector expressed as a protein, a recombinant microorganism transformed with the vector, and a soluble fusion protein antigen obtained from the recombinant microorganism as an active ingredient.

Foot and Mouth Disease virus (Foot and Mouth Disease virus) is a single-stranded bipolar RNA virus belonging to the genus Picornaviridae and Aphthovirus. It is a virus that exists in a capsid made of structural proteins without an envelope. It is classified into 7 serotypes, namely A, O, C, Asia1, SAT1, SAT2, and SAT3, and these major serotypes are divided into 200 different subtypes, and serotypes or serotypes vary well within serotypes, but serotypes. It is a disease that is very difficult to prevent and control disease occurrence because cross immunity between the liver is not achieved.

Currently, foot-and-mouth disease vaccine is widely used worldwide. However, first, foot-and-mouth disease inactivation vaccines are made using foot-and-mouth disease pathogens, so the risk remains. Due to vaccination, there is a possibility that the proliferation of the virus in the epithelial cells may occur, and there is also the possibility that the vaccinated livestock will remain a carrier state for actual foot-and-mouth disease virus infection in the future. Second, in line with the problem of direct use of foot-and-mouth disease virus, existing vaccines have a disadvantage in that a facility with a high level of quarantine is required in the manufacturing process. Viruses used in laboratories or vaccine manufacturing plants can spread outside and act as a source of infection, which can be a problem by itself, and it is difficult to easily determine vaccine production in clean countries where foot-and-mouth disease does not occur. Third, there is a disadvantage in that it is difficult to verify the efficacy or risk of the vaccine. In other words, it is always necessary to test animals in a shielded facility for risk verification, and it is difficult to keep the performance constant for each vaccine manufactured. Lastly, the existing vaccine is made by concentrating the supernatant of cells infected with foot-and-mouth disease virus, so it contains a large amount of non-structural protein. -antibody test can be difficult. Therefore, it is necessary to develop a next-generation vaccine against foot-and-mouth disease virus, and the vaccine is not made using the entire foot-and-mouth disease virus, so it does not require special facilities, it is possible to manufacture a vaccine with uniform performance, and can distinguish vaccinated animals from naturally infected animals. It should proceed in the direction that can be done.

Foot-and-mouth disease virus is difficult to produce vaccines because there are many strains between serotypes due to genetic complexity and diversity, as well as many strains within one serotype. In addition, vaccines for one serotype do not have protective immunity against other serotypes, and there are cases where there is a difference of up to 30% of the total genes in the gene sequence of two strains belonging to the same serotype. The main argument is that it should be applied to each type. Therefore, for foot-and-mouth disease vaccine, it is important to take a quick response to identify the virus serotype prevalent in the area around the outbreak and to manufacture the vaccine according to the serotype. To this end, it is necessary to improve the development and production method of the current foot-and-mouth disease inactivating vaccine. Therefore, it is necessary to develop another type of vaccine that can complement the problems of the existing inactivated vaccine and highlight its advantages. In other words, it is necessary to develop a foot-and-mouth disease vaccine that enables rapid reproduction according to serotypes, does not require special facilities, and is capable of producing vaccines with uniform performance.

To date, various studies for next-generation vaccines have been conducted internationally in order to overcome the shortcomings of existing inactivated vaccines. For example, a vaccine using a foot-and-mouth disease cDNA recombinant virus, VP1 purified from viral particles (Challa et al., Vaccine 25:3328-3337, 2007) bioengineered VP1, chemically synthesized VP1 peptide (Zhang et al., Virology journal 8:268, 2011), VP1 epitope peptide (Gao et al., Virologica Sinica 25:18-26, 2010), viral vector expressing VP1 epitope, bacterial vector, plant vector (Andrianova et al., Biochemistry 76 :339-346, 2011), DNA vaccine encoding VP1 epitope (Wang et al., Vaccine 26:5135-5144, 2008), VP1-VP4 capsid delivery or VP1 epitope delivery via replication-defective human adenovirus vector ( Lu et al., Vaccine 26:48-53, 2008) and the like are known. Among these next-generation vaccines being developed, efforts have been made to use as a vaccine by recombinantly mass-producing and purifying the major epitopes of foot-and-mouth disease antigens from E. coli (Xin et al., African Journal of Microbiology Research 5 :486-495, 2011). However, when expressed in E. coli, the development of a recombinant foot-and-mouth disease vaccine has been limited to date due to difficulties in purification and mass production due to expression in an insoluble form (insoluble form).

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 were studying to express the Multi-VP1e antigen protein in large quantities as the most basic step for the development of a recombinant foot-and-mouth disease vaccine, but the codon-modified form of the Multi-VP1e antigen protein was not aggregated in a specific host cell. It showed high solubility, and it was confirmed that the antibody-producing ability was similarly high to that of a commercialized vaccine, and 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 the polynucleotide.

Another object of the present invention is to provide an antigen expression system capable of expressing a large amount of the Multi-VP1e antigen protein in the form of a soluble protein and purifying it.

Another object of the present invention is to provide a vaccine for preventing foot-and-mouth disease virus infection containing the Multi-VP1e antigen protein as an active ingredient.

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

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

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

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

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 vaccine composition for preventing foot-and-mouth disease virus comprising the soluble Multi-VP1e antigen protein as an active ingredient.

The present invention also provides a method for enhancing immunity against foot-and-mouth disease virus, comprising administering the soluble Multi-VP1e antigen protein to a right medicament.

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

In addition, since it is not made using the entire foot-and-mouth disease virus, antigens can be manufactured in general laboratory facilities rather than special facilities, and there is no concern of infectivity, so antigens can be safely supplied.

The recombinant microorganism according to the present invention is economical because it can mass-produce Multi-VP1e antigen protein in the form of a soluble protein, and is effective in preventing the spread of foot-and-mouth disease because it can replace the antigen expressed within a short period of time for the foot-and-mouth disease virus serotype that is expected to be outbreak .

Figure 1 shows the amino acid sequences of two epitopes present in foot-and-mouth disease virus O1/Manisa, A/Pocheon, and Asia/Shamir VP1 proteins.
Figure 2 shows the expression vector of the multi-VP1e antigen protein of foot-and-mouth disease virus O1 / Manisa, A / Pocheon, Asia / Shamir.
3 shows the peptide sequences of O1/Manisa, A/Pocheon, and Asia/Shamir used for antibody production and ELISA.
FIG. 4 shows the purified foot-and-mouth disease virus Multi-VP1e antigen protein confirmed by Coomassie Brilliant Blue staining and Western blot using each antibody obtained using the peptide of FIG. 3.
Figure 5 is a mixture of a mixture of Multi-VP1e antigen protein and SEPPIC's ISA201, a commercial vaccine, and ISA201, respectively, to mice, after intramuscular administration, a serum collection schedule for confirming antibody production, and an experiment on infection with foot-and-mouth disease virus ASIA1 Shamir strain after administration This is a schematic diagram of the schedule.
FIG. 6 is a diagram showing a multi-VP1e (O1/Manisa, A/Pocheon, Asia/Shamir) administration group of a mouse experiment conducted according to the schedule of FIG. 5.
FIG. 7 shows serum from the experimental group administered with the multi-VP1e antigen protein and ISA201 mixture to mice, the positive control group administered with a commercial vaccine, and the negative control group administered only ISA201 according to the schedule of FIG. 5, and then each type of foot-and-mouth disease. It is a diagram confirming by dilution ratio that the antibody was generated in mouse serum at a high rate through ELISA performed by coating the virus VP1 peptide.
FIG. 8 is a diagram confirming for each type that the antibody in the serum used in FIG. 7 was generated using SP ELISA performed by coating an inactivated foot-and-mouth disease virus.
FIG. 9 is a high survival rate in the group administered with the Multi-VP1e antigen protein mixture against the foot-and-mouth disease virus ASIA1 Shamir strain infection after administration of the Multi-VP1e antigen protein mixture formulation, the commercialized vaccine, and ISA201 according to the schedule of FIG. 5 above. It is a figure confirming what is shown.
FIG. 10 is a diagram confirming by qRT-PCR that complete removal of the virus occurred from day 1 in the group administered with the Multi-VP1e antigen protein mixture after infection with the foot-and-mouth disease virus ASIA1 Shamir strain of FIG. 9.
FIG. 11 shows the activation of T-immune cells by administration of Multi-VP1e antigen protein through ELISPOT performed by stimulating splenocytes isolated from each mouse group with Multi-VP1e antigen protein and each VP1 peptide shown in FIG. 2. It is a degree that confirms that it was born.
Fig. 12 is a diagram showing the results of screening pigs to be used in the experiment using PrioCHECK FMDV SP ELISA.
Figure 13 is, after screening 8-week-old pigs rearing on the farm through SP ELISA, serum was collected to confirm antibody production from each experimental group administered with a multi-VP1e antigen protein mixture by dose and a control group administered with ISA. This is a schematic diagram of the experiment schedule.
14 is a diagram showing the administration group of the pigs multi-VP1e (O1/Manisa, A/Pocheon, Asia Shamir) administered according to the schedule of FIG. 13.
FIG. 15 shows antibodies in serum by administration of a mixture of Multi-VP1e antigen protein through ELISA performed by diluting the serum of pigs collected by proceeding according to the schedule of FIG. 13 and coating each type of foot-and-mouth disease virus VP1 peptide. This is a diagram confirming that formation was made at a high rate even in pigs, which are the target animals.
FIG. 16 is a diagram confirming through SP ELISA for each type that antibodies were generated in the serum of pigs collected by proceeding according to the schedule of FIG. 13 above.
17 is a diagram showing the results of a pig screening test for verifying the efficacy of Multi-VP1e antigen protein.
FIG. 18 is a diagram schematically illustrating the schedule of experiments for infection with foot-and-mouth disease virus ASIA1 Shamir strain after administration of a Multi-VP1e antigen protein mixture formulation and a commercial vaccine, and ISA201 administration in pigs selected through SP ELISA from 8-week-old pigs rearing on a farm.
19 is a diagram showing changes in body temperature in pigs administered with Multi-VP1e (O1/Manisa, A/Pocheon, Asia Shamir) according to foot-and-mouth disease virus infection.
FIG. 20 shows that in the foot-and-mouth disease virus ASIA1 Shamir strain infection experiment conducted according to the schedule of FIG. 18, the clinical symptoms that appeared in each group were evaluated, and that the group administered with the Multi-VP1e antigen protein mixture did not show any clinical symptoms of viral infection. This is a confirmed degree.
Figure 21 is a diagram showing the amino acid sequence of the antigenic site of foot-and-mouth disease virus O/Jincheon, A/Pocheon, Asia/Shamir.
Figure 22 is a diagram showing a schematic diagram of recombination in a pHis parallel vector for the synthesis of Multi-VP1e antigen proteins expressing VP1 epitope of O/Jincheon, O/Pocheon, and ASIA1/Sharmir, which are the serotypes of the foot-and-mouth disease virus currently occurring.
FIG. 23 is a diagram confirming the multi-VP1e antigen protein purified using the gene of FIG. 21 by Coomassie Brilliant Blue staining and Western blot.
FIG. 24 is a diagram showing a multi-VP1e (O/Jincheon, A/Pocheon, Asia/Shamir) administration group of a mouse experiment conducted according to the schedule of FIG. 5.
FIG. 25 shows a high rate of antibody production in mouse serum through ELISA performed by collecting serum from mice administered with the purified Multi-VP1e antigen protein according to the schedule of FIG. 5 and then coating each type of foot-and-mouth disease virus VP1 peptide. This is a diagram confirming that it was made by dilution ratio.
Fig. 26 is a diagram of antibody production in the mouse serum shown in Fig. 25 as measured using SP ELISA against each type of foot-and-mouth disease virus.
Figure 27 is a diagram confirming that the group administered with the Multi-VP1e antigen protein against the foot-and-mouth disease virus ASIA1 Shamir strain infection to mice administered with the purified Multi-VP1e antigen protein and the commercial vaccine and ISA201 showed a high survival rate.
Fig. 28 is a diagram confirming that activation of T-immune cells has occurred by administration of Multi-VP1e antigen protein using a serotype of the foot-and-mouth disease virus currently popular using ELISPOT after separating mouse splenocytes of each group.
29 is a diagram showing the amino acid sequence of an antigenic site of foot-and-mouth disease virus O/Jincheon, O/Andong, and O/Manisa.
FIG. 30 is a diagram showing a schematic diagram of recombination in a pHis parallel vector for 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 peptide used in ELISA for the preparation of antibodies used to confirm the Multi-VP1e antigen protein of purified foot-and-mouth disease virus O/Jincheon, O/Andong, O/Manisa on SDS-PAGE and confirmation of the formed antibody It is a diagram showing the sequence for each type.
Figure 32 is a diagram confirmed by Western blot using each antibody obtained using the Coomassie Brilliant Blue staining method and the peptide of Figure 31 for the purified foot-and-mouth disease virus O/Jincheon, O/Andong, O/Manisa Multi-VP1e antigen protein. .
33 is performed by collecting serum from mice administered with Multi-VP1e antigen protein of foot-and-mouth disease virus O/Jincheon, O/Andong, and O/Manisa according to the schedule of FIG. 5, and then coating each type of foot-and-mouth disease virus VP1 peptide. It is a diagram confirming by dilution ratio that the antibody was generated at a high rate in mouse serum through the ELISA.
FIG. 34 is a diagram confirming that activation of T-immune cells occurred by administration of Multi-VP1e antigen protein of foot-and-mouth disease virus O/Jincheon, O/Andong, and O/Manisa after separating mouse splenocytes of each group using ELISPOT .
35 is a diagram showing the amino acid sequences of antigenic sites of foot-and-mouth disease virus A/Pocheon, A/Malay97, and A/Iraq.
36 is a diagram showing a schematic diagram of recombination in a pHis parallel vector for synthesis of Multi-VP1e antigen proteins expressing VP1 epitope of foot-and-mouth disease virus A/Pocheon, A/Malay97, and A/Iraq.
37 is a peptide used in ELISA for the preparation of antibodies used to confirm the Multi-VP1e antigen protein of purified foot-and-mouth disease virus A/Pocheon, A/Malay97, and A/Iraq by SDS-PAGE and for confirmation of the formed antibody. It is a diagram showing the sequence for each type.
FIG. 38 is a diagram confirming the purified foot-and-mouth disease virus A/Pocheon, A/Malay97, and A/Iraq Multi-VP1e antigen proteins by Coomassie Brilliant Blue staining and Western blot using each antibody obtained using the peptide of FIG. 37 .
Figure 39 is performed by collecting serum from mice administered with Multi-VP1e antigen protein of foot-and-mouth disease virus A/Pocheon, A/Malay97, and A/Iraq according to the schedule of Figure 5, and then coating each type of foot-and-mouth disease virus VP1 peptide. It is a diagram confirming by dilution ratio that the antibody was produced at a high rate in mouse serum through the ELISA.
FIG. 40 is a diagram confirming that activation of T-immunocytes occurred by administration of Multi-VP1e antigen protein of foot-and-mouth disease virus A/Pocheon, A/Malay97, and A/Iraq using ELISPOT after separating mouse splenocytes of each group. .
41 is a diagram showing the amino acid sequence of an antigenic site of foot-and-mouth disease virus Asia/Shamir, Asia/Mongolia05, and Asia/LC04.
42 is a diagram showing a schematic diagram of recombination in a pHis parallel vector for synthesis of Multi-VP1e antigen protein expressing VP1 epitope of foot-and-mouth disease virus Asia/Shamir, Asia/Mongolia05, Asia/LC04.
Figure 43 is a peptide used in ELISA for the production of antibodies used to confirm the Multi-VP1e antigen protein of purified foot-and-mouth disease virus Asia/Shamir, Asia/Mongolia05, Asia/LC04 by SDS-PAGE and confirmation of the formed antibody. It is a diagram showing the sequence for each type.
FIG. 44 is a diagram confirming the multi-VP1e antigen protein of purified foot-and-mouth disease virus Asia/Shamir, Asia/Mongolia05, Asia/LC04 by Coomassie Brilliant Blue staining and Western blot using each antibody obtained using the peptide of FIG. 43 .
45 is performed by collecting serum from mice administered with Multi-VP1e antigen protein of foot-and-mouth disease virus Asia/Shamir, Asia/Mongolia05, and Asia/LC04 according to the schedule of FIG. 5, and then coating each type of foot-and-mouth disease virus VP1 peptide. It is a diagram confirming by dilution ratio that the antibody was produced at a high rate in mouse serum through the ELISA.
46 is a diagram confirming that activation of T-immunocytes occurred by administration of Multi-VP1e antigen protein of foot-and-mouth disease virus Asia/Shamir, Asia/Mongolia05, Asia/LC04 after separating mouse splenocytes of each group using ELISPOT .

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

In the present invention, in the Multi-Vp1e antigen protein, RGD motifs and C-terminus epitopes according to three serotypes may be randomly arranged. For example, the Multi-VP1e antigen protein of the foot-and-mouth disease virus may be composed of the RGD motif and C-terminus of the foot-and-mouth disease virus VP1 protein in the form as shown in FIG. 1, but O1/Manisa, A/Pocheon, Asia/Shamir and Likewise, it is not limited to a specific serotype of foot-and-mouth disease virus, and all functional equivalents applied after being modified in a form containing the VP1 epitope for specific serotypes O/Jincheon, A/Pocheon, and Asia/Shamir in epidemics at the time as shown in FIG. Includes.

In the present invention, the term "functional equivalent" refers to a purified antigen protein comprising a VP1 epitope for various types of foot-and-mouth disease virus, and this is for at least one serotype VP1 epitope, preferably 3 serotypes or its It refers to various types of purified antigen proteins that can be synthesized and used for the above serotypes.

In the present invention, antigen epitopes of FMDV VP1 protein useful for constructing a recombinant protein are amino acid residues 132-162 and 192-212 of FMDV VP1 protein (SEQ ID NOs: 1 to 6, SEQ ID NOs: 9 to 14, SEQ ID NO: 17. To 22 and SEQ ID NOs: 33 to 40), but is not limited thereto.

That is, the Multi-VP1e antigen protein of the present invention includes various types of VP1 epitopes that exhibit immunity in the host in addition to the RGD motif and C-terminus, and includes variants according to each type of protein mutation according to the virus outbreak. In the present invention, the term "variant" means that one or more amino acid residues are deleted, inserted, non-conservative or conservative substitutions, or a combination of one or more amino acid residues in the amino acid sequence of the epitope of the VP1 protein expressed by a specific serotype according to the periodic prevalence of the virus do.

In a preferred embodiment of the antigen protein of the present invention, the randomly arranged epitopes of the recombinant Multi-VP1e antigen protein are linked to each other via a peptide linker. Peptide linkers between antigenic epitopes should not affect the linear structure of the antigenic epitopes. 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 shows that the "GP linker" does not affect the linear structure of the antigen epitope randomly arranged in the recombinant Multi-VP1e protein in the present invention, and the recombinant Multi-VP1e protein induces the production of protective antibodies in animals. It indicates that it can be used as a possible peptide linker.

In the present invention, the Multi-VP1e antigen protein may be characterized by having 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 the Multi-VP1e antigen protein.

A method for obtaining a polynucleotide encoding the Multi-VP1e antigen protein of the present invention is 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 serotype to be protected is obtained, a sequence encoding a peptide linker is added, and then they are ligated together to encode the entire Multi-VP1e antigen protein. It is possible to form polynucleotides.

According to another embodiment of the present invention, the polynucleotide is characterized in that it is modified to suit the codon usage frequency of E. coli. When a polynucleotide of a recombinant antigen is composed of a codon that is frequently used in Escherichia coli and expressed in Escherichia coli, the probability of expression as a soluble protein and the amount of expression are significantly 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). In addition, problems that may occur when protein expression using polynucleotides with a minor codon can be avoided. In other words, if a protein is expressed using a codon that is less frequently used in E. coli, the translation process may be interrupted or the sequence of nucleotides may be changed (frame shifting), and the amino acids cannot be properly structured (mis-incorporation of amino acids). Protein may not be expressed (Robinson, M. et al. Nucleic acids research 12, 6663-6671, 1984). In addition, when codons with a low frequency of use in E. coli are used, it may affect the growth of Escherichia coli and cause problems with the protein expression level (V Emilsson & CG Kurland EMBO Journal 9(13) , 4359-4366, 1990). ).

The codon-modified polynucleotide of the present invention may be composed of 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 the polynucleotide. Expression vectors, such as prokaryotic expression vectors or eukaryotic expression vectors, that can be used to introduce the polynucleotides of the present 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 the vector. Since the expression level and modification of the target protein are different depending on the recombinant microorganism (host cell), it is important to select and use 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 may preferably be Escherichia coli . As the E. coli strain of the present invention, for example, BL21-Codon Plus(DE3)-RIPL, BL21-Codon Plus(DE3)-RG, OverExpress™ C41(DE3) and C43(DE3) Competent Cells, Value 10 ⁷ HIT-21 , DH5α™ Competent Cells, etc. may be used, preferably BL21-Codon Plus (DE3)-RIPL.

The present inventors sought the BL21-Codon Plus(DE3)-RIPL, BL21-Codon Plus(DE3)-RG, OverExpress™ to find the E. coli host cell system in which the codon-modified Multi-VP1e antigen protein is expressed in a highly soluble form. C41(DE3) and C43(DE3) Competent Cells, Value 10 ⁷ HIT-21 and DH5α™ Competent Cells were tested by introducing various codon-modified polynucleotides. As a result, in the case of OverExpress™ C41(DE3) and C43(DE3) Competent Cells and Value 10 ⁷ HIT-21, DH5α™ Competent Cells, Multi-VP1e antigen protein was not expressed in a water-soluble form, and BL21-Codon Plus ( In the case of DE3)-RG cells, a small amount of Multi-VP1e antigen protein was expressed in a water-soluble manner, but it was expressed in a yield that was more than 10 times lower than that of BL21-Codon Plus (DE3)-RIPL cells, and finally BL21-Codon Plus (DE3). -It was confirmed that the RIPL cells express the soluble protein in the highest yield, and the present invention was completed.

The above results are thought to be due to the fact that BL21-Codon Plus (DE3)-RIPL cells can more efficiently express the Multi-VP1e antigen protein, including a gene that additionally expresses tRNA. In addition, BL21-Codon Plus(DE3)-RIPL cells express not only tRNA genes, but also genes such as argU, ileY, and LeuW, and they facilitate the expression of proteins in the AT- or GC-rich genome, thereby making the expression of VP1 proteins more efficient. It is believed that it can be mediated.

According to another embodiment of the present invention, the present invention provides a method for mass-producing a 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 E. coli with the vector;

(c) It may be a mass production method of a soluble Multi-VP1e antigen protein comprising the step of crushing the E. coli cultured in step (b) to obtain a Multi-VP1e antigen protein.

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

In a preliminary experiment, the present inventors have transformed BL21-Codon Plus(DE3)-RG, OverExpress™ C41(DE3) and C43(DE3) Competent Cells, Value 10 ⁷ HIT-21 transformed with a recombinant vector encoding Multi-VP1e antigen protein. , DH5α™ Competent Cells and BL21-Codon Plus (DE3)-RIPL for different E. coli, after inducing protein expression at 37°C and 20°C, respectively, electrophoresis was performed to determine the expression and solubility of the Multi-VP1e antigen protein. It was confirmed through. As a result, it was confirmed that the expression-induced Multi-VP1e antigen protein at 20°C has higher solubility than the expression-induced protein at 37°C.

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

In the present invention, the vaccine composition may further include a pharmaceutically acceptable carrier and/or adjuvant. Such suitable carriers and/or adjuvants are inorganic adjuvants, for example aluminum hydroxide or aluminum phosphate; As organic adjuvants, for example CpG DNA or poly A; Microorganisms and extracts thereof; And oil adjuvants, and the like, and the adjuvant used in the present invention is not limited to ISA201 or any specific type of adjuvant.

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

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 twice a day to the target animal. It can be administered by intramuscular injection.

The term "effective amount" as used herein means that the component of the vaccine is an amount sufficient to induce an immune response specific to FMDV in the vaccinated animal. The effective amount can be easily determined by a person 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, the present inventors have two epitopes of VP1 of three serotypes of foot-and-mouth disease virus O1/Manisa/Turkey/69, A/Pocheon/001/KOR/2010, ASIA/Shamir/89 (FIG. 1 : SEQ ID NO: 1-6) was synthesized in a pHis-parallel vector and purified to be water-soluble using the E. coli expression system. When the purified Multi-VP1e antigen protein was administered to a mouse, the formation of antibodies against each serotype was confirmed by ELISA. And SP ELISA, it was confirmed through ELIPOT that it is involved in the activation of T immune cells, and it was proved that a successful vaccine effect was shown by confirming a high survival rate and low proliferation of the virus against foot-and-mouth disease virus infection after administration (Fig. 4). -11).

In addition, the present inventors synthesized and purified current serotypes O/Jincheon, A/Pocheon, and ASIA1/Sharmir (FIG. 21: SEQ ID NO: 9-14) in expression vectors in addition to the three types of serotypes used in the above experiment. As a result of inoculating mice with one Multi-VP1e antigen protein in the same way and confirming, Multi-VP1e antigen protein consisting of different types of serotypes is also effective as a vaccine against foot-and-mouth disease virus infection through antibody formation in serum and activation of T immune cells. It was confirmed that represents (Figs. 21-28).

In addition, the present inventors confirmed that the present invention can be used as a vaccine having an intensive effect against a specific type of virus by synthesizing a VP1 multiepitope against a specific type of virus in an expression vector to express the Multi-VP1e antigen protein. Foot-and-mouth disease virus O type O/Jincheon, O/Andong, O/Manisa (FIG. 29: SEQ ID NO: 17-22), foot-and-mouth disease virus A type A/Pocheon, A/Malay97, A/Iraq (FIG. 35: SEQ ID NO: 25 -30), foot-and-mouth disease virus Asia type Asia/Shamir, Asia/Mongolia05, Asia/LC04 (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 an experiment using an antigen protein, antibody formation in mouse serum and activation of T immune cells were confirmed (FIGS. 29-46).

In order to confirm the efficacy of Multi-VP1e antigen protein in pigs, which are target animals for foot-and-mouth disease virus, the present inventors administered Multi-VP1e antigen protein to pigs selected through SP ELISA on a farm, and then separated serum and separated ELISA and SP ELISA. Using Multi-VP1e antigen protein administration, antibody formation was confirmed, and Multi-VP1e antigen protein was administered to pigs selected by the same method, infected with foot-and-mouth disease virus, and clinical symptoms of foot-and-mouth disease virus after infection were evaluated. The vaccine effect by the antigen protein was demonstrated (Figs. 12-20).

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

Hereinafter, the present invention will be described in detail by examples. However, the following examples are intended to illustrate the present invention to make it easier to understand, and the contents of the present invention are not limited by the examples.

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

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

The linker of each site was linked using a linker amino acid sequence (GPGPG) to create a gene sequence, and the multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) gene (SEQ ID NO: 7) was synthesized by gene synthesis. It was named as.

Example 2. Construction of His tag-Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) E. coli expression vector

The synthesized Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) gene was modified by inserting EcoRI at the 5'end and XhoI at the 3'end using chain polymerization (PCR). After ligation to T Easy Vector (Invitrogen, Korea), digestion with restriction enzymes inserted into 5'and 3'was ligated back to the pHis parallel vector. The produced His tag-Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) plasmid was transformed and amplified in E. coli DH5a, and the amplified plasmid protein was expressed in E. coli (E. coli BL21-CodonPlus(). DE3)-RIPL Competent Cells (Agilent technology, #230280)) were transformed to induce the target protein to be expressed (Fig. 2).

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

E. coli BL21-CodonPlus(DE3)-RIPL Competent Cells (Agilent technology, #230280) transformed with His tag-Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) plasmid LB containing ampicillin The plate was streaked using 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 then transferred to 3 L of LB broth for main culture. In the main culture, when _{the value of OD 600} was about 0.6, the final concentration of IPTG was put into 1 mM. The lactose operator was stimulated using IPTG to induce protein formation, followed by incubation at 37°C for 4 hours. E. coli only was obtained by centrifugation, washed with a buffer solution (50 mM Tris-HCl, 1 mM EDTA, pH 8.0), resuspended with the same buffer solution, and then the target protein from E. coli using sonication. Was eluted, and separated using a centrifuge (12,000 rpm/30min/4°C), and then only the supernatant was obtained to obtain a water-soluble protein. The degree of expression of this water-soluble protein fraction was confirmed by Coomassie Brilliant Blue staining. The identified water-soluble His tag-Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein was purified using FPLC and IMAC to purify only the target protein antigen.

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

The purified target protein Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein was electrophoresed by 12% SDS-PAGE, and confirmed using Coomassie Brilliant Blue staining. In addition, it was confirmed using Western blot. The antibody used at this time was an antibody prepared using a peptide (FIG. 3) containing an RGD motif in VP1 of each serotype O1 manisa, A pocheon, and Asia1 shamir. These antibodies were reacted by mixing with PBST at a ratio of 1:1000 each in Western blot, and antigen proteins were identified at about 25 Kda, the size of which His tag (3Kda) and Multi-VP1e (20Kda) antigen proteins were combined ( Fig. 4).

Example 5. Preparation of vaccine by mixing antigen protein and immunity enhancer

This experiment was conducted using ISA201 provided by SEPPIC, confirming that it elicited the best immunity enhancing effect through previous experiments. The antigen protein Multi-VP1e protein and the adjuvant were mixed at a weight ratio of 1:1, and a 1ml syringe and a 3 way stopcock were used. After mixing according to the method recommended by SEPPIC, it was stored at room temperature for 1 hour and then stored at 4°C for one day before use.

Example 6. Analysis of immune response induction after FMDV vaccination in mice

Mice were used by purchasing 5-week-old female C57BL/6N crljori from Oriental Bio (Korea). The inoculation plan of the multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein mixed with the adjuvant was performed as shown in FIG. 5, and the groups of mice were divided as shown in FIG. 6. The experiment was conducted with three experimental mice. Each experimental group was divided into 1) a group administered with a mixture of ISA 201 and PBS buffer, 2) a group administered with a combination of foot-and-mouth disease polyvalent antigen protein (Multi-VP1e) and ISA 201, and 3) a group administered with a commercially available tetravalent vaccine. Thus, the efficacy of the foot-and-mouth disease polyvalent antigen protein vaccine was tested.

FMDV Type SP Antibody ELISA Kit (Life technology)를 각 혈청형 별로 수행하였다 (도 8). Each experimental group was separated by 13 animals, and group 1) was mixed with 40 ul of PBS and 40 ul of ISA201, and 40 ul of Multi-VP1e and 40 ul of ISA201 were mixed in group 1) and injected intramuscularly twice at an interval of 1 week. Group 3) was injected intramuscularly with 100ul of a commercial vaccine in the same manner as used in the foot-and-mouth disease diagnosis department of the Agriculture, Forestry and Livestock Quarantine Headquarters. Antibodies at OD450nm through ELISA carried out by coating with antigen peptides against O1 manisa, A pocheon, and Asia1 shamir or with Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) protein using the serum obtained on the 21st day thereafter. The production rate was analyzed (Fig. 7). In addition, PrioCHECK￠ which is performed by coating inactivated FMDV for more accurate analysis.

FMDV Type SP Antibody ELISA Kit (Life technology) was performed for each serotype (FIG. 8).

FMDV Type Antibody ELISA 분석 결과 Multi-VP1e(O/Manisa-A/Pocheon-Asia/Shamir)와 ISA 201 혼합제가 상용화 백신과 유사하거나 더 높은 항체 생성을 유도하는 것을 확인하였다.Analysis results and PrioCHECK￠ at OD 450nm

As a result of FMDV Type Antibody ELISA analysis, it was confirmed that the combination of Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) and ISA 201 induces the production of antibodies similar to or higher than those of commercial vaccines.

Example 7. Analysis of efficacy of vaccines against FMDV infection

Two weeks after the mice were finally inoculated with Multi-VP1e antigen protein, an _{amount of FMDV Asia1 shamir virus 10LD 50} was administered intraperitoneally to induce infection. The virus used in the mouse virus infection test was serotype of Asia1/shamir/89, which was carried out in the shielding room of the foot-and-mouth disease diagnosis department of the Agricultural and Livestock Quarantine Service. The amount of virus was used as _{10LC 50.}

As a result of analyzing the survival rate for 8 days after infection, all of the control mice died within 3 days, and both the Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) and ISA 201 mixed drug administration group and the commercialized vaccine administration group survived ( Fig. 9).

As a result of measuring the amount of virus by qRT-PCR using mouse serum samples obtained on the 1st and 3rd days after FMDV Asia1 shamir virus infection, the virus was completely removed from the 3rd day in the commercial vaccine-treated group, whereas the multi-VP1e polyvalent antigen protein-treated group was 1 It was confirmed that complete removal occurred from the first day (FIG. 10). Through this, it was confirmed that the vaccine efficacy of the antigen protein Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) was excellent.

Example 8. Analysis of cytokine production in mouse splenocytes

Isolation of splenocytes in the spleen of mice inoculated with Multi-VP1e antigen protein to determine whether T immune cells responded to the antigenic protein of Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) and participated in the immune function. Then, Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) protein, O1/Manisa/Turkey/69, A/Pocheon/001/KOR/2010 and Asia1/Shamir/89, etc. T-immune cell response experiments were conducted using a stimulation method.

The experiment was conducted using the ^{BD TM ELISPOT set.} First, purified IFN-r antibody and purified IL-4 antibody were diluted 1:200 in PBS, dispensed at 100 ul/well, and coated. The next day, the spleen was separated to obtain splenocytes, and red blood cells were removed using ACK lysis buffer, and then washed with RPMI media and resuspended. The number of cells in the media was counted, and splenocytes obtained from each individual ^{were equally adjusted at a concentration of 7x10 5} /100ul, and then dispensed at 100ul/well. The stimulant (VP1 peptide) was equally diluted with RPMI media (10% FBS) at a concentration of 3 ug/100 ul, and then dispensed at 100 ul/well. After storing in an incubator at 37° C. and 5% CO ₂ for 24 hours, the detection antibody and Streptabidin-HRP were reacted. Finally, the color was developed using an AEC buffer, and the number of dots generated by IFN-r and IL-4 was measured and expressed as a graph (FIG. 11). It was confirmed that the experiment was carried out correctly through Mitogen stimulator (1 ug/well) as a positive control and media only as a negative control, and IFN-r and IL-4 were produced only in the vaccine group, and the formation of spots due to color development was confirmed. . Through the above experiment, it was confirmed that the Multi-VP1e antigen protein contributed to the activation immune function of T immune cells against the Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein.

Example 9. Immunogenicity analysis after inoculation of Multi-VP1e antigen protein in pigs

FMDV Type O SP Atibody ELISA Kit를 이용하여 선별검사를 수행하였다(도 12). 선별검사 결과 높은 PI(percentage inhibition)값이 나온 돼지를 제외하여 12마리를 선별하고, 3마리씩 4가지 그룹으로 나누었다. 면역증강제와 PBS버퍼의 혼합물을 투여하여 대조군을 설정하였고, Multi-VP1e(O/Manisa-A/Pocheon-Asia/Shamir) 항원단백질을 100ug, 200ug, 300ug으로 세 가지 용량을 설정하여 3가지 실험군을 설정하였다(도 14).PrioCHECK￠ targeting the serum of 20 8-week-old pigs raised on the farm

Screening was performed using the FMDV Type O SP Atibody ELISA Kit (Fig. 12). As a result of the screening test, 12 pigs were selected, excluding pigs with high PI (percentage inhibition) values, and they were divided into 4 groups of 3 each. A control group was set by administering a mixture of an adjuvant and a PBS buffer, and three different doses were set for the Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein: 100 ug, 200 ug, and 300 ug. Was set (Fig. 14).

According to the administration schedule of FIG. 13, inoculation of Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein was performed at week 0 and week 4, and blood collection was performed at week 3 and week 6. Pig blood was stored at 4° C. for 1 hour and then serum was obtained using a centrifuge. Antigen coating ELISA was performed using pig serum at 6 weeks. Serum was sequentially diluted from a ratio of 1:50 to 2 times, and the dilution factor was set up to 1:1600.

The vaccine group showed high antibody values against all antigens such as Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein and O1 manisa, A pocheon, and VP1 peptide of Asia1 shamir virus. Also showed a high antibody value (Fig. 15). Pig serum was subjected to each serotype of O, A, and Asia using PrioCHECK® FMDV Type SP Antibody ELISA. As a result of ELISA after the first and second inoculations, it was confirmed that the multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein inoculation group showed a high value, and the individual difference was also reduced (FIG. 16). .

Example 10. Analysis of vaccine efficacy of Multi-VP1e antigen protein in pigs

A screening test was performed using the PrioCHECK® FMDV Type O SP Antibody ELISA Kit for the serum of 50 pigs of 8 weeks of age raised on the farm (FIG. 17). As a result of the screening test, 11 pigs were selected excluding pigs with high PI values, and an experiment plan was established as shown in FIG. 18. Inoculation of commercial vaccine and Multi-VP1e (O/Manisa-A/Pocheon-sia/Shamir) antigen protein was performed at week 0 and week 4, and at the time of 5 week, Asia 1 type Shamir strain was infected.

As shown in FIG. 19, as a result of monitoring the change in body temperature for 9 days after infection, the increase in body temperature of the commercial vaccine group and the Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein inoculation group was not confirmed. And as a result of confirming clinical symptoms such as blister formation in the mouth or feet, loss of appetite, limbs, and inability to stand for 10 days after inoculation with Asia 1 type Shamir strain, as shown in Fig. 20, the clinical symptoms shown in the Adjuvant only group are commercialized. It was not seen in the vaccine group and in the Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein inoculation group.

According to the results of this experiment, the vaccine candidate prepared by mixing 300 ug of Multi-VP1e (O/Manisa-A/Pocheon-Asia/Shamir) antigen protein with ISA201 VG protects against FMDV vaccination not only in the mouse model but also in the target animal pig. It was confirmed that it can induce the immunity that can do it.

Examples 11-14. Protective effect against various foot-and-mouth disease virus subtypes

Example 11. Confirmation of efficacy against new foot-and-mouth disease outbreaks

11-1. Production of Multi-VP1e antigen protein of a new foot-and-mouth disease development strain

A vaccine that can be used to prevent foot-and-mouth disease can be produced by verifying the efficacy of a vaccine by producing a new Multi-VP1e as an epitope within VP1 of the foot-and-mouth disease virus currently occurring. Among the serotypes of Example 1, O1 manisa was replaced with O/Jincheon, which is the O type serotype that is currently occurring, and the GH loop of the three serotypes O/Jincheon, A/Pocheon, Asia1/Shamir (VP1 amino acid 132 to 162) and C-terminus (VP1 amino acid 192 to 212) were determined (Fig. 21), and a gene was produced, which was multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir ) It was named as a gene.

11-2. His tag-Multi-VP1e(O/Jincheon-A/Pocheon-Asia/Shamir) E. coli expression vector production, target protein expression and purification

A chain polymerization reaction (PCR) was performed by inserting EcoRI at the 5'end and an XhoI site at the 3'end to make His tag-JC-Multi-VP1 plasmid as in Example 2 and E. coli BL21-CodonPlus(DE3)-RIPL It was transformed into Competent Cells (Agilent technology, #230280) (Agilent technology) to induce the target protein to be expressed. The water-soluble target protein was isolated from E. coli through the method of Example 3 and the expression level 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. Confirmation of purified His tag-Multi-VP1e(O/Jincheon-A/Pocheon-Asia/Shamir) protein

The purified target protein, Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) antigen protein, was confirmed by Kumisi Brilliant Blue staining as in Example 4, and RGD in A VP1 and Asia1 VP1 used in Example 4 Expression of the antigen protein was confirmed through Western blot using the motif antibody (FIG. 23).

11-4. Confirmation of immunogenicity against foot-and-mouth disease antigen in mice

As in Example 5, Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) protein and an immune adjuvant were mixed in a weight ratio of 1:1, and the mouse groups were divided as shown in FIG. 24, and the inoculation plan proceeded as shown in FIG. I did. As in Example 6, serum was obtained from each mouse on the 21st day after inoculation, and coated with antigen peptides against Ojincheon, A pocheon, and Asia1 shamir, or with Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) protein. The antibody production rate was analyzed at OD450nm through an ELISA (FIG. 25). In addition, Type SP Antibody ELISA Kit (Life technology) was performed for each serotype (FIG. 26). As a result of the two antibody production assays, it was confirmed that the mixture of Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) and ISA 201 induces high antibody production similar to those of commercial vaccines.

11-5. Analysis of vaccine efficacy of antigen protein

Mice were inoculated with Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) antigen protein 2 weeks after the last day of inoculation with FMDV Asia1 shamir virus 10LD ₅₀ intraperitoneally to induce infection. As a result of analyzing the survival rate for 8 days after infection, all of the control mice died on the second day, and only one in the group administered with Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) and the ISA 201 combination drug died, and the group receiving the commercialized vaccine. All survived (FIG. 27). Through this, it was confirmed that the vaccine efficacy of the Multi-VP1e antigen protein is excellent.

11-6. Analysis of cytokine production in mouse splenocytes

104 Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) Multi-VP1e (O/Jincheon-A/Pocheon -Asia/Shamir) spleen cells in the spleen of mice inoculated with antigen protein were isolated, and Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) protein, O/Jincheon, A/Pocheon/001/KOR/ In 2010, ELISPOT was conducted using Asia1/Shamir/89. As a result of measuring the secretion of IFN-r and IL-4, it was confirmed that IFN-r and IL-4 were produced only in the vaccine group (FIG. 28). Through the above experiment, Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) and O jincheon, A pocheon, Asia1 shamir antigen by Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) antigen protein. It was confirmed that it contributed to the activation immune function of T immune cells against the protein. Through this experiment, it was confirmed that it is possible to produce a vaccine that can be used to prevent foot-and-mouth disease by applying a new Multi-VP1e according to the foot-and-mouth disease virus occurring at the time.

Example 12. Confirmation of vaccine efficacy against foot-and-mouth disease virus O type

12-1. Production of Multi-VP1e antigen protein of foot-and-mouth disease virus O type

A vaccine that can be used to prevent foot-and-mouth disease can be produced by verifying the efficacy of a vaccine by producing a new Multi-VP1e with the VP1 epitope of a specific type of foot-and-mouth disease virus. O/Jincheon, O/Andong, O/Manisa Three types of foot-and-mouth disease virus O-type GH loop (VP1 amino acid 132-162) and C-terminus (VP1 amino acid 192-212) were determined and antigenic sites were determined (Fig. 29), a gene was produced and named as Multi-VP1e (O/Jincheon, O/Andong, O/Manisa) gene.

12-2. His tag-Multi-VP1e (O/Jincheon, O/Andong, O/Manisa) E. coli expression vector production, target protein expression, purification and confirmation

A chain polymerization reaction (PCR) was performed by inserting EcoRI at the 5'end and an XhoI site at the 3'end to make His tag-JC-Multi-VP1 plasmid, and E. coli BL21-CodonPlus(DE3)-RIPL Competent Cells (Agilent technology , #230280) was induced so that the target protein could be expressed. Through the method of Example 3, the water-soluble target protein was isolated from E. coli and the level of expression was confirmed. The identified water-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 confirmed by Coomassie brilliant blue staining as in Example 4, and O/Jincheon, O/Andong, O/Manisa VP1 Using the inner RGD motif antibody (Fig. 31), the expression of the antigen protein was confirmed through Western blot (Fig. 32).

12-3. Immunogenicity analysis for foot-and-mouth disease antigen in mice

As in Example 5, Multi-VP1e (O/Jincheon, O/Andong, O/Manisa) protein and an immune adjuvant were mixed in a weight ratio of 1:1, and the experiment was conducted in the same manner as in the schedule of FIG. 5. As in Example 6, serum was obtained from each mouse on the 21st day after the start of inoculation and used as antigenic peptides or Multi-VP1e (O/Jincheon, O/Andong, O/Manisa) proteins against O/Jincheon, O/Andong, and O/Manisa. The antibody production rate was analyzed at OD450nm through ELISA performed by coating (FIG. 33). As a result of antibody production analysis, it was confirmed that the mixture of Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) and ISA 201 produced a lot of antibodies similar to commercial vaccines. In addition, by analyzing the response of T immune cells in the same manner as in Example 8, it was confirmed that the immune response of specific T immune cells was induced by Multi-VP1e (O/Jincheon-A/Pocheon-Asia/Shamir) (Fig. 34).

Example 13. Confirmation of vaccine efficacy against foot-and-mouth disease virus A type

13-1. Production of Multi-VP1e antigen protein of foot-and-mouth disease virus A type

A/Pocheon, A/Malay97, A/Iraq Three types of foot-and-mouth disease virus O-type GH loop (VP1 amino acid 132-162) and C-terminus (VP1 amino acid 192-212) were determined and antigenic sites were determined (Fig. 35), a gene was produced. This was named Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) gene.

13-2. His tag-Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) E. coli expression vector production, target protein expression, purification and confirmation

A chain polymerization reaction (PCR) was performed by inserting EcoRI at the 5'end and an XhoI site at the 3'end to make His tag-JC-Multi-VP1 plasmid, and E. coli BL21-CodonPlus(DE3)-RIPL Competent Cells (Agilent technology , #230280) was induced so that the target protein could be expressed. Through the method of Example 3, the water-soluble target protein was isolated from E. coli and the level of expression was confirmed. The identified water-soluble His tag-Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) antigen protein was purified using FPLC and IMAC. The purified target protein Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) antigen protein was confirmed by Coomassie brilliant blue staining as in Example 4, and A/Pocheon, A/Malay97, A/Iraq VP1 Using the inner RGD motif antibody (FIG. 37), the expression of the antigen protein was confirmed through Western blot (FIG. 38).

13-3. Immunogenicity analysis for foot-and-mouth disease antigen in mice

As in Example 5, Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) protein and an immune adjuvant were mixed at a weight ratio of 1:1, and the experiment was conducted according to the schedule of FIG. 4. As in Example 6, serum was obtained from each mouse on the 21st day after inoculation, and the antigenic peptide against A/Pocheon, A/Malay97, A/Iraq or Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) protein was used. The antibody production rate was analyzed at OD450nm through ELISA performed by coating (FIG. 39). As a result of antibody production analysis, it was confirmed that the combination of Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) and ISA 201 induces high antibody production similar to those of commercial vaccines. In addition, by analyzing the response of T immune cells in the same manner as in Example 8, it was confirmed that the immune response of specific T immune cells was induced by Multi-VP1e (A/Pocheon, A/Malay97, A/Iraq) (FIG. 40 )

Example 14. Confirmation of vaccine efficacy against foot-and-mouth disease virus Asia type

14-1. Production of Multi-VP1e antigen protein of the foot-and-mouth disease virus Asia type

Asia/Shamir, Asia/Mongolia05, Asia/LC04 Three types of foot-and-mouth disease virus Asia-type GH loop (VP1 amino acid 132-162) and C-terminus (VP1 amino acid 192-212) were determined and antigenic sites were determined (Fig. 41), a gene was produced, and it was named Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) gene.

14-2. His tag-Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) E. coli expression vector production, target protein expression, purification and confirmation

A chain polymerization reaction (PCR) was performed by inserting EcoRI at the 5'end and an XhoI site at the 3'end to make His tag-JC-Multi-VP1 plasmid, and E. coli BL21-CodonPlus(DE3)-RIPL Competent Cells (Agilent technology , #230280) was induced so that the target protein could be expressed. Through the method of Example 3, the water-soluble target protein was isolated from E. coli and the level of expression was confirmed. The identified water-soluble His tag-Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) antigen protein was purified using FPLC and IMAC. The purified target protein Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) antigen protein was confirmed by Coomassie Brilliant Blue staining as in Example 4, Asia/Shamir, Asia/Mongolia05, Asia/LC04 VP1 Using the inner RGD motif antibody (Fig. 43), the expression of the antigen protein was confirmed through Western blot (Fig. 44).

14-3. Immunogenicity analysis for foot-and-mouth disease antigen in mice

As in Example 5, Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) protein and an immune adjuvant were mixed at a weight ratio of 1:1, and the experiment was conducted according to the schedule of FIG. 4. As in Example 6, serum was obtained from each mouse on the 21st day after the start of inoculation, and as an antigen peptide against Asia/Shamir, Asia/Mongolia05, Asia/LC04, or Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) protein. The antibody production rate was analyzed at OD450nm through ELISA performed by coating (FIG. 45). As a result of antibody production analysis, it was confirmed that the mixture of Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) and ISA 201 induces high antibody production similar to those of commercial vaccines. In addition, by analyzing the response of T immune cells in the same manner as in Example 8, it was confirmed that the immune response of specific T immune cells was induced by Multi-VP1e (Asia/Shamir, Asia/Mongolia05, Asia/LC04) (Fig. 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 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> <223> 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> <223> 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 Ile Ala Pro Ala Lys Gly Pro Gly Pro Gly Tyr Asn Gly Lys 115 120 125 Thr Ala Tyr Gly Glu Thr Thr Ser Arg 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 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 Pro 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 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> <223> 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> <223> 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 Ala Pro Ala Lys Gly Pro Gly Pro Gly Tyr Asn Gly Lys Thr 115 120 125 Ala Tyr Gly Glu Thr Thr Ser Arg 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 Pro 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> <223> 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> <223> 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 Pro 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> <223> 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> <223> 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 Ile Ala 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 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 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 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 Arg 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> <223> Polynucleotide of Multi-VP1e (Asia/Shamir, Asia/Mongolia05, 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> <223> Synthetic protein Multi-VP1e (Asia/Shamir, Asia/Mongolia05, 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 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 Arg Gly Asp Leu Ala Ala Leu Ala Gln Arg Val Ser Arg 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 Pro Gly Gly Gly Gly Pro 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 (15)

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: 25 to 30, The soluble Multi-VP1e antigen protein of No. 25 to 30 is linked via a peptide linker of the Gly-Pro-Gly-Pro-Gly sequence (SEQ ID NO: 41), respectively. 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. A polynucleotide encoding the soluble Multi-VP1e antigen protein of any of claims 1 or 2. 4. The polynucleotide of claim 3, wherein the polynucleotide is modified to match the codon usage of E. coli. The polynucleotide according to claim 4, which is a nucleotide sequence of SEQ ID NO: 31. A recombinant vector comprising the polynucleotide of claim 5. A recombinant microorganism transformed with the vector of claim 6. 8. The recombinant microorganism according to claim 7, wherein the microorganism is E. coli. 9. The recombinant microorganism according to claim 8, wherein the E. coli is BL21-Codon Plus (DE3) -RIPL cells. (a) preparing a recombinant vector comprising the polynucleotide of claim 3;
(b) transforming and culturing Escherichia coli with the vector;
(c) disrupting E. coli cultured in step (b) to obtain a Multi-VP1e antigen protein. 11. The method of mass production according to claim 10, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 31. 11. The method of mass production according to claim 10, 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 claim 1 or 2 as an active ingredient. 14. The vaccine composition of claim 13, further comprising a pharmaceutically acceptable carrier or adjuvant. 11. A method for promoting immunity against foot-and-mouth disease virus comprising administering the soluble Multi-VP1e antigen protein of claim 1 or 2 to a fungus.

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