KR101566371B1 - Recombinant foot and mouth disease virus expressing protective antigen of Asia1 type standard vaccine strain and producing method thereof - Google Patents

Abstract

The present invention relates to a recombinant foot-and-mouth disease virus expressing a protective antigen of a standard vaccine virus of foot-and-mouth disease type 1 serotype and a method for producing the same. The recombinant foot-and-mouth disease virus according to the present invention has an excellent effect of expressing an epitope of VP1 protein or VP1 protein of As1 / Shamir / 89, and thus can be effectively used for prevention of foot-and-mouth disease including foot-and-mouth disease type 1.

Description

Technical Field [0001] The present invention relates to recombinant foot-and-mouth disease viruses expressing protective antigens of standard vaccine viruses of foot-and-mouth disease type 1 and methods for producing the same,

The present invention relates to a recombinant foot-and-mouth disease virus expressing a protective antigen of a standard vaccine virus of foot-and-mouth disease type 1 serotype and a method for producing the same.

Foot-and-mouth disease (FMD) is a virus-like disease that infects animals with split hoofs and has a fast disease-spreading power. Foot-and-mouth disease is classified as a disease that can be spread across countries by the World Animal Health Organization (OIE), and it is a very important disease because of its economic loss. Because of these characteristics, the occurrence of foot and mouth disease in livestock trade among countries is considered to be a very important check factor. Foot and mouth disease pathogens are a single stranded bipolar RNA virus, and are known to belong to the genus Piconaviridae and Aphthovirus. In addition, foot-and-mouth disease is classified according to seven different serotypes (A, O, C, Asia1, SAT1, SAT2, SAT3).

There are more than 8 different types of foot-and-mouth disease virus O type. If strictly developed, vaccines for these local types should be developed and used as vaccines. However, for the development of all the vaccine strains, the outbreaks must be continuously cultured in cells to have adequate propagation capacity. However, this method is time consuming and there is no guarantee that the results will be good.

In addition, foot-and-mouth disease virus

It is a pathogen that shows genetic origin. Many researchers, including several times,

It is internationally contraindicated to be used as an attenuated vaccine, which has a reduced pathogenicity to foot-and-mouth disease virus to date, because sex is maintained.

The use of attenuated virus against foot-and-mouth disease has a problem of restoration of virulence and it is difficult to distinguish it from outbreaks of foot-and-mouth disease virus using live monsoon vaccine.

Therefore, using the existing vaccine virus, the vaccine strain needed can be rapidly

It should be made in a form that can be produced in a timely manner so that it can be developed and used.

On the other hand, foot-and-mouth disease virus type O Manisa is known to be the most widely used vaccine virus in O-type vaccine, and it is possible to protect against viruses in ME-SA (including the Middle East-South Asia region type and PanAsia region type) In addition to the O type, Asian 1 and A type occur in the periphery, and the Asian 1 serotype can be classified into the group I-VI. Although the above-mentioned Asian 1 serotype occurs only in the Asian region, it has not only spread the virus in Turkey and Greece in 2000, but also recently, an increase in the incidence of the Asian 1 serotype has been reported.

Recently, it has been complicated in the East Asian region, and Afghanistan,

, Iran, Malaysia, Nepal, Pakistan and Thailand.

From the end of 2004 to the year of 2006, the Asian serotype 1 virus occurred in the autonomous regions of China, Myanmar, Mongolia, East Russia, Tajikistan and Vietnam.

Since 2005, the Asian one serotype V genome in East Asia, including China, has been identified as a genetically closely related virus with the isolated virus 25 years ago in India, and the Asian 1 virus in Bahrain in 2009 Which is estimated to come from. In particular, in 2007, North Korea's occurrence of Asia-type 1 was shocking. Since genotype V is generated around Korea, development of a vaccine against these viruses is urgently required.

In order to adequately defend against foot-and-mouth disease type 1 serotype, the inventors of the present invention found that P1 gene of Asian type 1 genotype V virus (Asia1 / MOG / 2005) substituted or inserted with As1 / Shamir / Was replaced with the P1 gene of foot-and-mouth disease virus O manisa vaccine to complete the recombinant foot-and-mouth disease virus of the present invention.

Korean Patent No. KR 10-1236203

PLoS one, Vol.8, Issue.1, e55228 (2013)

Therefore, an object of the present invention is to provide a recombinant vector comprising a gene of a foot-and-mouth disease type O virus that expresses the VP1 protein of Asia / Shamir / 89 or an epitope thereof, and a recombinant foot-and-mouth disease virus produced therefrom.

It is another object of the present invention to provide a recombinant vector and a method for producing the recombinant virus.

Still another object of the present invention is to provide a vaccine composition for preventing FMD virus comprising the recombinant FMDV as an active ingredient.

Still another object of the present invention is to provide a method for preventing foot-and-mouth disease using the vaccine composition.

The present invention relates to a method for the prevention and treatment of foot-and-mouth disease, wherein the P1 gene of the foot-and-mouth disease type O virus is substituted with the P1 gene of the Asian 1 serotype V type virus and the VP1 site of the substituted P1 gene is replaced with the VP1 gene of Asia / Shamir / A recombinant vector comprising the inserted gene of the foot-and-mouth disease type O virus.

Also provided are recombinant foot-and-mouth viruses produced by said vectors.

In addition,

1) inserting the gene of foot-and-mouth disease type O virus into a recombinant vector;

2) replacing the P1 gene of foot-and-mouth disease type O virus with the P1 gene of Asian 1 serotype type V virus in the recombinant vector of step 1); And

3) substituting or inserting the VP1 site of the P1 gene of the recombination vector of step 2) with the VP1 gene of As1 / Shamir / 89 or a partial nucleotide sequence thereof.

Also, the present invention provides a method for preparing recombinant FMDV comprising introducing a recombinant vector produced by the above method into a cell to proliferate.

Hereinafter, the present invention will be described in detail.

The VP1 gene of As1 / Shamir / 89 of the present invention is characterized by being represented by the nucleotide sequence of SEQ ID NO: 11. The base sequence is a nucleotide sequence encoding an epitope, preferably a 140-160th amino acid sequence of the VP1 protein.

The As1 / Shamir / 89 foot-and-mouth disease virus is a standard for foot-and-mouth disease type 1 vaccine, and has a defense effect against the type-I foot-and-mouth disease virus.

The gene of the foot-and-mouth disease type O virus is characterized by being a whole gene of foot-and-mouth disease type O vaccine strain Manisa. In the entire gene region, 3B1 region can be substituted and 3B2 region deleted in 3B1 region, 3B2 region and 3B3 region. The substitution site of the 3B1 region of the gene is 22 to 36 bp of the 3B1 region represented by the nucleotide sequence of SEQ ID NO: 1 and the deletion site of the 3B2 region of the gene is a 37 to 105 bp region of the 3B2 region represented by the nucleotide sequence of SEQ ID NO: 2 But is not limited thereto.

In the present invention, "foot-and-mouth disease virus O-type Manisa strain" is the most widely used vaccine virus in O-type vaccine, and it is possible to protect the virus against ME-SA (including Middle East-South Asian type and PanAsia type) It is known.

The genotype of the Asian 1 serotype virus can be divided into Groups I to VI, and in the present invention, the Asian 1 serotype genotype V virus is preferable, and more preferably the Asia 1 / MOG / 2005 virus. The entire sequence of the Asia1 / MOG / 2005 virus is registered with GeneBank approval number EF614458.1.

The recombinant vector of the present invention may be a pOm3B-AsM-ShamVP1 vector represented by the cleaved map of Fig. 3, and the recombinant vector may be represented by the nucleotide sequence of SEQ ID NO: 14. In addition, the recombinant vector of the present invention may be pOm3B-AsM-ShamVP1epi represented by the cleavage map of Fig. 4, and may be represented by the nucleotide sequence of SEQ ID NO: 15.

As used herein, the term "vector" means a DNA product containing a DNA sequence operably linked to an appropriate regulatory sequence capable of expressing the DNA in the appropriate host. The vector may be a plasmid, phage particle or simply a potential genome insert. Once transformed into the appropriate host, the vector may replicate and function independently of the host genome, or, in some cases, integrate into the genome itself. Because the plasmid is the most commonly used form of the current vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purpose of the present invention, it is preferable to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) a cloning start point that allows replication to be efficiently made to include several hundred plasmid vectors per host cell, (b) a host cell transformed with the plasmid vector And (c) a restriction enzyme cleavage site into which the foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site is not present, using a synthetic oligonucleotide adapter or a linker according to a conventional method can easily ligate the vector and the foreign DNA. The recombinant vector of the present invention and the recombinant foot-and-mouth disease virus can be produced by conventional gene manipulation and transformation methods, and a virus having a small amount can be obtained in an appropriate amount in a continuous passage through cell culture.

In the present invention, "P1 gene" is a nucleotide sequence encoding a P1 protein and may include VP1, VP2, VP3 and VP4 regions. In the present invention, the region includes a nucleotide sequence encoding a protein, It is used as a concept involving sequences and genes.

The cells of the present invention can be used for the treatment of cancers, feline animals, wild boar and animals, bovine animals, deer and animals, giraffe and animals, fecalities and animals, camelids, hippopotamus, horses and animals, (ZZ-R) and hamster kidney cells (BHK-21), black goat kidney cells (BGK), pigs (pigs), and the like. (IBRS-2) and small intestine cells (LF-BK). However, the present invention is not limited thereto.

The present invention provides a vaccine composition for preventing foot-and-mouth disease comprising the recombinant FMDV as an active ingredient.

The recombinant foot-and-mouth disease virus of the present invention is excellent in the effect of expressing the VP1 protein or its epitope of the standard vaccine of Asia type 1 foot-and-mouth disease, and thus can be effectively used for prevention of foot-and-mouth disease including foot-and-mouth disease type 1.

The vaccine may include, but is not limited to, a live vaccine, an attenuated vaccine, or a vaccine. The foot-and-mouth disease is preferably Asian 1 type foot-and-mouth disease, but is not limited thereto.

The composition of the present invention may contain one or more known active ingredients which are excellent in preventing foot-and-mouth disease infections together with recombinant foot-and-mouth disease virus.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, sucrose solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, Other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added and formulated into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) depending on the desired method, and the dose may vary depending on the weight, age, sex, , Diet, administration time, method of administration, excretion rate, and severity of the disease. The dose of the recombinant FMDV is preferably about 1 × 10 ⁰ to 1 × 10 ²⁰ cells / animal, preferably about 1 × 10 ¹ to 1 × 10 ¹⁵ cells / animal.

In addition, the recombinant foot-and-mouth disease virus of the present invention is produced by manipulating a gene part of the foot-and-mouth disease virus O Manisa and weakens the pathogenicity to animals. Therefore, the recombinant foot-and- Can be used, and the virus titer can be effectively protected against virus localization occurring in Korea, East Asia, China or Taiwan, without significantly differing from the original virus.

In addition, the present invention provides a method for preventing foot-and-mouth disease comprising administering a recombinant foot-and-mouth disease virus to a subject.

Wherein the object is at least one selected from the group consisting of wild boars and animals, bovine animals, deer and animals, giraffes and animals, females and animals, camelids, hippopotamus, rhinoceroses and animals.

The recombinant foot-and-mouth disease virus according to the present invention has an excellent effect of expressing an epitope of VP1 protein or VP1 protein of As1 / Shamir / 89, and thus can be effectively used for prevention of foot-and-mouth disease including foot-and-mouth disease type 1.

Brief Description of the Drawings Fig. 1 is a schematic diagram showing the cloning of the whole gene of the foot-and-mouth disease type O vaccine Manisa and the insertion of the P1 gene of the Asian one serotype virus into the plasmid.
FIG. 2 is a diagram showing a genomic diagram of recombinant FMDV expressing the defensive antigen of foot-and-mouth disease type 1 serotype vaccine of the present invention.
FIG. 3 is a schematic diagram of a recombinant plasmid (pOm3B-AsM-ShamVP1) into which a VP1 defense protein gene of foot-and-mouth disease type 1 serotype vaccine standard is inserted.
4 shows a schematic diagram of a recombinant plasmid (pOm3B-AsM-ShamVP1epi) in which a nucleotide sequence encoding a VP1 epitope (amino acid at 140-160) of foot-and-mouth disease type 1 serotype vaccine is inserted.
FIG. 5 is a graph showing the cytopathic effect of the recombinant foot-and-mouth disease virus-infected cells of the present invention. FIG.
FIG. 6 is a graph showing the virus titer after the recombinant foot-and-mouth disease virus of the present invention is proliferated in a hamster cell line (BHK-21) and then infected with a fetal tongue cell line (ZZ-R).

Hereinafter, the contents of the present invention will be described in detail through experimental examples. However, the following experimental examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited thereto.

[ Example  1] Recombinant plasmid pO - Manisa  And pom - AsMOG - P1 Manufacturing

1. Recombinant plasmid pO - Manisa  Produce

The recombinant plasmid pO-Manisa was prepared by amplifying the whole genome of the foot-and-mouth disease O type Manisa virus (GenBank Accession No. AY593823.1) by PCR and inserting the O-Manisa gene into a plasmid (pBluescript SK II).

More specifically, the PCR amplification was carried out in the following manner.

In general, after preparing cDNA for O-Manisa virus using a random primer used in cDNA synthesis, a specific primer (GenBank Accession No. AY593823.1, which can be amplified based on O-Manisa virus gene information using the above cDNA, Corresponding to 20 mers of each of the 5'- and 3'- genes as the bases) were prepared and PCR was performed.

PCR was performed at 98 ° C for 30 seconds at 98 ° C using 5X buffer (FINNZYMES, 10 μl), 10 mM dNTPs (1 μl), Phusion enzyme (2 U / μl, 0.5 μl) and sterile distilled water (35.5 μl) After 25 cycles of 10 seconds, 65 ° C for 30 seconds, and 72 ° C for 2 minutes and 30 seconds, the reaction was carried out at 72 ° C for 10 minutes, and the PCR product was inserted into a plasmid (pBluescript SK II) pO-Manisa.

2. Recombinant plasmids pom - Manisa Manufacturing of -3B2d

A plasmid containing O-Manisa (GenBank Accession No. AY593823.1) prepared in 1-1 above was inactivated by in vitro mutagenesis in a commonly used in vitro mutagenesis method to replace some 3B region (Recombinant plasmid pO-Manisa-3B2d (position 22-36 bp in the 3B region, gagcgtcagaaacct; SEQ ID NO: 1) and deletion (37-105 bps site in the 3B region, ctgagagtga aaaccaagtt gccacaacag gagggaccct acgctggccc gatggataga cagaaaccg; Application 10-2012-0152084, December 24, 2012).

3. Recombinant plasmid pom - AsMOG - P1  Produce

Among the recombinant plasmids (pO-Manisa-3B2d) prepared in Example 1-2, the gene coding for the P1 protein as a defense antigen was designated as Asia1 / MOG / 2005 virus (GenBank Accession No. (POm-AsMOG-P1) into which a recombinant gene was inserted, was prepared by substituting the gene coding for the P1 protein, which is a protective antigen of EF614458.1. The gene encoding the P1 protein was substituted as follows. MOG / 2005 virus (GenBank Accession No. EF614458.1) using the forward primer (5'-GGAGCCGGGCAATCCAGTCCGGCGACC-3 ') of SEQ ID NO: 3 and the reverse primer (5'-CTGTTTCTCAGGTGCAATGATCTCCTG- Were amplified by PCR.

At this time, the PCR was performed as follows. After 30 seconds at 98 占 폚 at a volume of 5X buffer (FINNZYMES, 10 占 퐇), 10mM dNTPs (1 占 퐇), Phusion enzyme (2U / 占 퐇, 0.5 占 퐇 and sterilized distilled water (35.5 占 퐇) PCR was carried out at 25 cycles at 65 ° C for 30 seconds and 72 ° C for 60 seconds, followed by final reaction at 72 ° C for 10 minutes.

Using the forward primer (5'-CTTCTAAATTTTGACCTGCTCAAATTGACCTGCTCAAATTGGCGGG-3 ') of SEQ ID NO: 5 and the reverse primer (5'-CTTGAGCCTTTTCTGGACCTTTGTTTTCCAC-3') of SEQ ID NO: 6, the P1 gene PCR was carried out under the above-described conditions.

Thereafter, a TAKARA Long Ligation kit was performed with the amplified P1 gene, and it was finally confirmed that cloning (pOm-AsMOG-P1) was properly performed through analysis of the entire nucleotide sequence (pOm-AsMOG-P1, Application 10-2013-0073428, June 26, 2016).

FIG. 1 shows a schematic diagram (pOm-AsMOG-P1) of a step of cloning the whole gene of the foot-and-mouth disease type O vaccine Manisa and inserting the Asian 1 serotype P1 defense protein gene into the plasmid.

< Example  2> VP1  or VP1  140-160 Preparation of Recombinant Foot-and Mouth Virus Replaced with Amino Acid Sites.

1. The present invention pOm3B - Asm - ShamVP1  Plasmid preparation.

The primer for VP1 amplification of the vaccine standard host As1 / Shamir / 89 based on the plasmid (pOm-AsMOG-P1) prepared in Example 1 contained the forward primer of SEQ ID NO: 7 (5'-ACCACCACCACTGGCGAATCAGCAGAT-3 ' (5'-CTTCTAAATTTTGACCTGCTCAAATTGGCGGG-3 ') of SEQ ID NO: 9 and the reverse primer of SEQ ID NO: 10 (5'-CTGTTGGCGGGCATCCACCGGCAGTCG -3 '), the pOm3B-AsM-ShamVP1 plasmid of the present invention was prepared by ligating the VP1 gene of As1 / Shamir / 89 (shown in SEQ ID NO: 11) with pOm-AsMOG-P1. A process for producing the recombinant plasmid of the present invention is shown in Fig. 2, and a schematic diagram of plasmid pOm3B-AsM-ShamVP1 (the entire sequence is shown in SEQ ID NO: 14) is shown in Fig.

As shown in FIG. 3, it was confirmed that the VP1 site of pOm-AsMOG-P1 was substituted with the VP1 site of As1 / Shamir / 89.

2. The present invention pOm3B - Asm - ShamVP1epi  Plasmid production

In order to insert the nucleotide sequence coding for the amino acid sequence 140-160 of the VP1 gene of As1 / Shamir / 89, the nucleotide sequence encoding the 140-160 amino acid sequence, which is an epitope of VP1, was used as a primer for amplification. The VP1 epitope was amplified using the forward primer (5'-GCCCTCGCACAAAGGTTGCGCAGAAGGTTGCGCAGAAGGTTGCGCAGAGTGAACAACC-3 ') and the reverse primer (5'-CGCCATGTCGCCGCGCCTTGCGAGGGCGGCAAG-3') of SEQ ID NO: 13 and inserted into pOm-AsMOG- -SharmVP1epi (the entire sequence is shown in SEQ ID NO: 15). A schematic diagram thereof is shown in Fig.

As shown in FIG. 4, it was confirmed that a nucleotide sequence encoding the 140 to 160 amino acid sequence of VP1 was inserted into pOm-AsMOG-P1.

3. Production of recombinant foot-and-mouth disease virus using the plasmid of the present invention

The recombinant foot-and-mouth disease virus was recovered by preparing the recombinant plasmid (pOm3B-AsM-ShamVP1 or pOm3B-AsM-ShamVP1epi) with a restriction enzyme to prepare a single fragment of the gene. BHKT7-9 cells (a cell line expressing T7 RNA polymerase ) Was transformed with a lipofectamine transfection reagent (Life Technologies) and cultured for 2 to 3 days to obtain a recombinant virus. Thereafter, the obtained virus was inoculated through a continuous passage in ZZ-R (fetal tongue) cells or BHK-21 (young hamster kidney) cells to proliferate the virus. Thereafter, the cytopathic effect is shown in Fig. In addition, the viral titers of the recombinant foot-and-mouth disease virus of the present invention after proliferation in the hamster cell line (BHK-21) and infection with the goat fetal tongue cell line (ZZ-R) are shown in FIG.

As shown in FIG. 5, it was confirmed that the recombinant virus was reshaped in the cells infected with foot-and-mouth disease virus of the present invention, and the virus infection could be detected by the fluorescent antibody method, and it was confirmed that there was an effect of denaturing cells.

As shown in FIG. 6, when the foot-and-mouth disease virus of the present invention was infected with the fetal tongue cell line (ZZ-R), it was confirmed that the virus titer was high.

<110> Animal and plant quaratine agency <120> Recombinant foot and mouth disease virus expressing protective          antigen of Asia 1 type standard vaccine strain and producing          method thereof <130> P1-52 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> it is DNA sequence of substitution region in 3B of O-Manisa <400> 1 gagcgtcaga aacct 15 <210> 2 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> it is DNA sequence of deletion region in 3B of O-Manisa <400> 2 ctgagagtga aaaccaagtt gccacaacag gagggaccct acgctggccc gatggataga 60 cagaaaccg 69 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for P1 / MOG / 2005 virus <400> 3 ggagccgggc aatccagtcc ggcgacc 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for P1 of Asia 1 / MOG / 2005 virus <400> 4 ctgtttctca ggtgcaatga tctcctg 27 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for P1-deleted-O-manisa virus <400> 5 cttctaaatt ttgacctgct caaattggcg gg 32 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for P1-deleted-O-manisa virus <400> 6 cttgagcctt ttctggacct ttgttttcca c 31 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for VP1 of As1 / Shamir / 89 virus <400> 7 accaccacca ctggcgaatc agcagat 27 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for VP1 of As1 / Shamir / 89 virus <400> 8 ctgcttctca ggtgcaatga tctcc 25 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for pOm-AsMOG-P1 vector <400> 9 cttctaaatt ttgacctgct caaattggcg gg 32 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for pOm-AsMOG-P1 vector <400> 10 ctgttggcgg gcatccaccg gcagtcg 27 <210> 11 <211> 582 <212> DNA <213> Artificial Sequence <220> <223> it is VP1 DNA sequence of As1 / Shamir / 89 virus <400> 11 tatggaggag agactcagac agccagacgg cttcacactg acgtcgcctt cattcttgac 60 aggtttgtga aactcactgc tcccaagaac atccaaaccc tcgatctcat gcagatcccc 120 tcacacacgc tggttggagc actacttcgt tctgcgacgt actacttctc agacctggag 180 gtcgcgcttg tccacacagg cccggtcacc tgggtgccca acggcgcgcc caaggatgct 240 ctaaacaacc agaccaaccc aactgcctat cagaagcaac ccatcacccg cctggcactc 300 ccctacaccg ccccccatcg tgtgctggca acagtgtaca acgggaagac ggcgtacggg 360 gaaacgacct caaggcgcgg cgacatggcg gccctcgcac aaaggttgag cgctcggctg 420 cccacctcct tcaactacgg cgccgtgaag gccgacacca tcactgagct tttgatccgc 480 atgaagcgcg cggagacata ttgccctagg cctttactag cccttgacac cactcaggac 540 cgccgcaaac aggagatcat tgcacctgag aagcaggttt tg 582 <210> 12 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> it is forward primer for VP1 protein 's epitope of As1 / Shamir / 89          virus <400> 12 gccctcgcac aaaggttgcg cagagtgaac aacc 34 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> it is reverse primer for VP1 protein epitope of As1 / Shamir / 89          virus <400> 13 cgccatgtcg ccgcgccttg cgagggcggc aag 33 <210> 14 <211> 11019 <212> DNA <213> Artificial Sequence <220> <223> it is full DNA sequence of pOm3B-AsM-SharmVP1 <400> 14 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa agggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagttggact cacggcaccg 780 cgtggccatt ttagctgggt tgtgcggacg aacactgctt gcgcatctcg cgtgaccggt 840 tagtactctt accactatcc gcctacttgg tcgttagcgc tgtcctgggc actcttgttg 900 ggggctgttc aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 cccccccccc ccccctaggt tttaccgtcg ttcccgacgt taatggggaa acaaccacaa 1080 gcttaacacc gtcttgcccg acgtaaaagg gctgcaacca aaaagcttgt gccgcctttc 1140 ccggcgttaa tgggaggtaa ccacaagaca aaccttcacc cggaagtaaa acggcaactt 1200 cacacagttt tgcccgtttt cgtgagaaat gggccgtcaa cgcacgaaac gcgccgtcgc 1260 ttgaggagga cttgtacaaa cacgatctat gcaggtttcc acaactgaca caaaccgtgc 1320 aacttgaaac cccgcctggt ctttccaggt ctagaggggc gacattttgt actgtgcttg 1380 actccacgct cggtccacta gcgagtgtta gtagtagcac tgttgcttcg tagcggagca 1440 tgatggccgt gggagcttcc ccttggtaac aaggacccac ggggccaaaa gccacgtcct 1500 accggaccca tcatgtgtgc aaacccagca cggcaacttt actgcgaaaa ccactttaag 1560 gtgacactga tactggtact caatcactgg tgacaggcta aggatgccct tcaggtaccc 1620 cgaggtaaca cgcgacactc gggatctgag aaggggactg gggcttcttt aaaagtgccc 1680 agtttaaaaa gcttctatgc ctgaataggc gaccggaggc cggcgccttt tcactgtttt 1740 actactgttt tcatgaatac aactgactgt ttcaccgccc tgttacacgc tctcagagag 1800 atcaaaacac tgtttctttt acggacacaa ggaaagatgg aattcacact ttacaacggt 1860 gagaagaaaa ccttctactc cagacccaac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggtatgt tgatgagcct ttctttgact gggtctacga ctcgcctgaa 1980 aacctcactc ttgaggcaat caaacagttg gaagagacaa ccggtcttga gctgcacgag 2040 ggtggaccac ccgctctcgt catctggaac atcaaacact tgcttcacac cggaatcggc 2100 actgcctcac gccctagcga ggtgtgtatg gtggacggaa cggacatgtg tttagctgat 2160 tttcatgctg gcattttcct gaaaggacag gaacatgctg tgttcgcctg tgtcacctcc 2220 aacgggtggt acgcgattga tgacgaggac ttttaccctt ggacaccgga cccgtccgac 2280 gttctggtgt ttgtcccgta cgatcaagaa ccgcttaacg gagagtggaa aacaaaggtc 2340 cagaaaaggc tcaagggagc cgggcaatcc agtccggcga ccgggtcgca gaaccagtca 2400 ggcaacactg gaagcatcat taacaactac tacatgcagc agtaccagaa ctccatggac 2460 acgcaacttg gagataacgc tatcagcgga ggctccaacg agggttccac ggacaccaca 2520 tccacacaca caaacaacac ccaaaacaat gattggttct cacgcttggc cagttcggcc 2580 tttagcggac tgtttggtgc tcttttggct gacaagaaaa cggaggagac aactctgctt 2640 gaagaccgca tactcaccac cagaaatggc cacacgacgt cgacgacaca gtcgagtgtt 2700 ggcgtaacat atggttacgc tgtggctgag gacgcggtat ctgggcctaa cacctcaggc 2760 ctggagaccc gcgtgacaca ggctgaacgg ttcttcaaga aacacctgtt tgactggacg 2820 ccggatttgt catttggaca ctgtcactac ttggaactcc cctctgaaca caagggcgtg 2880 tttggcagcc tcatgagctc ttatgcttac atgaggaacg ggtgggacat tgaggtgacc 2940 gctgttggaa atcagttcaa tggtggttgt ctcctcgtcg cactcgtgcc ggagctgaaa 3000 gagctcgaca cgcggcagaa gtatcagtta accctcttcc cacaccagtt cattaacccg 3060 cgcactaaca tgacggctca cattaacgtg ccgtacgtgg gtgtcaacag gtacgaccag 3120 tacgagctcc acaaaccgtg gacgcttgtg gtgatggtgg tggccccgct taccgtcaaa 3180 actggtggtt ctgaacagat caaggtttac atgaatgcag cgccgaccta cgtgcacgtg 3240 gcaggagaac tgccctcgaa agaggggata gttcctgtgg cgtgtgtgga cggttacggc 3300 aacatggtaa ccacggaccc gaagacggct gaccccgtct acgggaaagt gtctaacccc 3360 cccagaacaa gcttccctgg gcgcttcaca aacttccttg atgtagcgga ggcgtgtcca 3420 accttcctcc gcttcggaga agtaccattt gtgaagacgg tgaactctgg tgaccgcttg 3480 cttgccaagt ttgacgtgtc cctcgctgcg gggcacatgt ccaacaccta cttggcaggt 3540 ttggcacagt actacacaca gtacagcggc actatgaata tccacttcat gttcactgga 3600 cccacggatg ccaaagcccg ctacatggtg gcttacatac ctcctggtat gacaccgcca 3660 acggacccgg agcgggctgc acactgcatt cattctgagt gggacactgg actcaattct 3720 aaatttacct tttctatccc ttacctttct gctgcagact atgcttacac tgcttctgac 3780 gtggctgaga ccacgagtgt gcagggatgg gtgtgtattt accagatcac ccacggtaaa 3840 gctgaaggtg acgcgctggt cgtgtccgtc agcgctggca aggactttga gtttcgactg 3900 ccggtggatg cccgccaaca gaccaccacc actggcgaat cagcagatcc agtcacaacc 3960 acggttgaga actatggagg agagactcag acagccagac ggcttcacac tgacgtcgcc 4020 ttcattcttg acaggtttgt gaaactcact gctcccaaga acatccaaac cctcgatctc 4080 atgcagatcc cctcacacac gctggttgga gcactacttc gttctgcgac gtactacttc 4140 tcagacctgg aggtcgcgct tgtccacaca ggcccggtca cctgggtgcc caacggcgcg 4200 cccaaggatg ctctaaacaa ccagaccaac ccaactgcct atcagaagca acccatcacc 4260 cgcctggcac tcccctacac cgccccccat cgtgtgctgg caacagtgta caacgggaag 4320 acggcgtacg gggaaacgac ctcaaggcgc ggcgacatgg cggccctcgc acaaaggttg 4380 agcgctcggc tgcccacctc cttcaactac ggcgccgtga aggccgacac catcactgag 4440 cttttgatcc gcatgaagcg cgcggagaca tattgcccta ggcctttact agcccttgac 4500 accactcagg accgccgcaa acaggagatc attgcacctg agaagcagct tctaaatttt 4560 gacctgctca aattggcggg agatgtggag tccaaccctg ggcccttctt cttctccgac 4620 gtcaggtcaa atttctcaaa actggtagaa accatcaatc agatgcagga ggacatgtca 4680 acaaaacacg ggcctgactt taaccggttg gtgtccgcat ttgaggaatt ggccactgga 4740 gtgaaggcta tcagggccgg tctcgacgag gccaaaccct ggtacaaact catcaagctc 4800 ctgagccgct tgtcgtgcat ggccgctgta gcagcacggt caaaggaccc agtccttgtg 4860 gccatcatgc tggctgacac cggtcttgag attctggaca gcacctttgt cgtgaagaag 4920 atctccgact cgctctccag tctctttcac gtgccggccc ccgtcttcag tttcggagcc 4980 ccgattctgt tggccgggtt ggtcaaagtc gcctcgagtt tcttccggtc cacacccgaa 5040 gaccttgaga gagcagaaaa acagctcaaa gcacgtgaca ttaacgacat attcgccatt 5100 ctcaagaacg gcgagtggct ggtcaagctg atccttgcca tccgcgactg gatcaaagcg 5160 tggatcgcct cagaagaaaa gtttgtcacc atgacggact tggtgcctgg tatccttgaa 5220 aagcagcggg atctcaacga cccgagtaag tacaaggaag ccaaggagtg gctcgacaac 5280 gcgcgccagg cgtgtttgaa gagcgggaac gttcacattg ccaatttgtg caaagtggtc 5340 gccccggcac ccagcaagtc gagacccgaa cccgtggtcg tttgcctccg cggcaaatcc 5400 ggccagggga agagtttcct tgcgaacgtg ctcgcgcaag caatctccac ccacttcacc 5460 ggcagaactg attcggtttg gtactgcccg cctgaccctg accacttcga cggttacaac 5520 cagcagaccg ttgtcgtgat ggacgatttg ggccagaacc ccgatggcaa ggacttcaag 5580 tacttcgccc agatggtttc gaccacgggg ttcatcccgc ccatggcctc gcttgaggac 5640 aaaggcaagc ctttcaacag caaagtcatc attgctacca ccaacctgta ctcgggtttc 5700 accccgagaa caatggtgtg tcctgacgcg ctgaaccgga ggttccactt tgacatcgac 5760 gtgagtgcca aggacgggta caaagttaac aacaaattgg acataatcaa agctcttgaa 5820 gacacccaca ccaacccagt ggcgatgttc caatacgact gtgcccttct aaacggtatg 5880 gcagttgaaa tgaagagaat gcaacaggat atgttcaagc ctcaaccacc cctccagaac 5940 gtgtaccaac tcgttcacga ggtgattgaa cgggtcgagc tccacgagaa ggtgtcgagc 6000 cacccgattt tcaaacagat atcaattcct tcccaaaagt ctgtgttgta cttcctcatt 6060 gagaaaggcc aacacgaagc agcaattgaa ttctttgagg gaatggtgca tgactccatc 6120 aaggaagagc tccggcccct catccaacag acctcatttg tgaaacgcgc ttttaagcgc 6180 ctgaaggaaa actttgagac tgttgccctg tgtttgactc ttttggcaaa catagtgatc 6240 atgatccgcg agactcgcaa gagacaacag atggtggacg atgcagtgaa tgactacatt 6300 gagaaggcaa acatcaccac agatgacaag actcttgacg aggcggaaaa gaaccctcta 6360 gagaccagcg gtgccagcac tattggtttc agagagagaa ctctcccggg gcacaaggcg 6420 agcgatgacg tgagctccga gcccgccaaa cccgtggagg accgaccaca agctgaaggg 6480 ccctacgccg gaccacttaa gaaaccagtg gcattgaaag tgagagcaag agccccggtc 6540 gtgaaggagg gaccctacga gggaccggtg aagaagcctg tcgctttgaa agtgaaagcc 6600 aagaacttga ttgtcactga gagtggtgcc ccaccgaccg acttgcagaa gatggtcatg 6660 ggcaacacta agcctgttga gctcatcctc gacgggaaga cggtagccat ctgctgtgct 6720 accggagtgt ttggcactgc ctacctcgta cctcgtcacc tcttcgcgga gaagtacgac 6780 aagataatgt tggacggtag agccatgaca gacagtgact acagagtgtt tgagtttgag 6840 attaaagtaa aaggacagga catgctctca gacgctgcac tcatggtgct tcaccgtggg 6900 aaccgcgtga gagacatcac gaaacatttt cgtgacacag caagaatgaa gaaaggcacc 6960 cccgttgtcg gtgtgatcaa caacgccgac gttgggagac tgattttctc tggagaggcc 7020 cttacctaca aagacattgt agtgtgcatg gatggagaca ccatgccggg cctgtttgcc 7080 tacagagccg ccaccaaggc tggttactgc gggggagccg ttctcgccaa ggacggagcc 7140 gacacattca tcgttggcac ccactccgca ggtggtaacg gagttggata ctgctcgtgc 7200 gtgtccaggt ccatgctcct gaaaatgaag gcacacattg accctgaacc acaccacgag 7260 gggttgattg ttgataccag agatgtggaa gagcgcgtgc atgtcatgcg taaaaccaag 7320 cttgcaccca ccgtggcaca cggtgtgttt aaccctgaat ttggtcccgc tgccttgtcc 7380 aacaaggacc cgcggctgaa cgaaggggtt gtcctcgatg aagtcatctt ctccaaacac 7440 aagggagaca cgaaaatgtc tgaggaggac aaagcgctgt tccgccgctg cgctgccgac 7500 tacgcgtcgc acttgcacag cgtgctgggg acggcaaatg ccccattgag catctatgag 7560 gccatcaaag gcgtcgacgg gctcgatgcc atggagccgg acaccgcgcc cggcctcccc 7620 tgggccctcc aggggaaacg ccgtggtgcg ttgattgact tcgagaacgg cacggtcgga 7680 cccgaagtcg aggctgccct aaagctcatg gagaaaagag agtacaaatt tgcttgccag 7740 accttcctga aagacgagat tcgtccgatg gaaaaagtac gtgctggcaa gactcgcatt 7800 gtcgacgttt tgcccgtgga acacattctt tacaccagga tgatgattgg cagattctgt 7860 gctcaaatgc acacaaacaa tggaccgcag attggctcag cggtcggttg caatcctgat 7920 gttgattggc aaagatttgg cacacatttt gctcagtaca gaaacgtgtg ggatgtggac 7980 tattcggcct ttgatgctaa ccactgcagt gacgcaatga acatcatgtt tgaggaggta 8040 tttcgcacag acttcggttt ccacccaaat gctgagtgga ttctgaagac tcttgtgaac 8100 acggagcacg cctatgagaa caaacgtatc actgttgagg gcgggatgcc gtctggctgt 8160 tccgcgacaa gcatcatcaa cacaattttg aacaacattt atgtgctcta cgctcttcgt 8220 agacactatg agggagttga gctggacacc tacaccatga tctcctacgg agatgacatc 8280 gtggttgcaa gtgactacga tctggatttt gaggctctca aaccccactt caaatctctt 8340 ggtcaaacca tcactccagc tgacaaaagc gacaaaggtt ttgttcttgg tcactccatt 8400 accgatgtca ctttcctcaa aagacacttc cacatggact atggaactgg gttttacaaa 8460 cctgtgatgg cctcaaagac cctcgaggcc attctctcct ttgcacgccg tgggaccata 8520 caggagaagt tgatctccgt ggcaggactc gccgtccact caggacctga cgagtaccgg 8580 cgtctctttg agcccttcca gggtctcttc gagattccaa gctacagatc actttacctg 8640 cgttgggtga acgccgtgtg cggtgacgca taatccctca gatgtcacaa ttggcagaaa 8700 gactctgagg cgagcgacgc cgtaggagtg aaaagcccga aagggctttt cccgcttcct 8760 attccaaaaa aaaaaaaaaa aaactagttc tagagcggcc gccaccgcgg tggagctcca 8820 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt 8880 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 8940 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 9000 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 9060 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 9120 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 9180 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 9240 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 9300 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 9360 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 9420 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 9480 gt; ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 9600 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 9660 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 9720 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 9780 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 9840 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 9900 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 9960 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 10020 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 10080 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 10140 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 10200 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 10260 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 10320 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 10380 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 10440 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 10500 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 10560 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 10620 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 10680 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 10740 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 10800 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 10860 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 10920 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 10980 aaataggggt tccgcgcaca tttccccgaa aagtgccac 11019 <210> 15 <211> 11055 <212> DNA <213> Artificial Sequence <220> <223> it is full DNA sequence of pOm3B-AsM-SharmVP1epi <400> 15 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa agggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagttggact cacggcaccg 780 cgtggccatt ttagctgggt tgtgcggacg aacactgctt gcgcatctcg cgtgaccggt 840 tagtactctt accactatcc gcctacttgg tcgttagcgc tgtcctgggc actcttgttg 900 ggggctgttc aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 cccccccccc ccccctaggt tttaccgtcg ttcccgacgt taatggggaa acaaccacaa 1080 gcttaacacc gtcttgcccg acgtaaaagg gctgcaacca aaaagcttgt gccgcctttc 1140 ccggcgttaa tgggaggtaa ccacaagaca aaccttcacc cggaagtaaa acggcaactt 1200 cacacagttt tgcccgtttt cgtgagaaat gggccgtcaa cgcacgaaac gcgccgtcgc 1260 ttgaggagga cttgtacaaa cacgatctat gcaggtttcc acaactgaca caaaccgtgc 1320 aacttgaaac cccgcctggt ctttccaggt ctagaggggc gacattttgt actgtgcttg 1380 actccacgct cggtccacta gcgagtgtta gtagtagcac tgttgcttcg tagcggagca 1440 tgatggccgt gggagcttcc ccttggtaac aaggacccac ggggccaaaa gccacgtcct 1500 accggaccca tcatgtgtgc aaacccagca cggcaacttt actgcgaaaa ccactttaag 1560 gtgacactga tactggtact caatcactgg tgacaggcta aggatgccct tcaggtaccc 1620 cgaggtaaca cgcgacactc gggatctgag aaggggactg gggcttcttt aaaagtgccc 1680 agtttaaaaa gcttctatgc ctgaataggc gaccggaggc cggcgccttt tcactgtttt 1740 actactgttt tcatgaatac aactgactgt ttcaccgccc tgttacacgc tctcagagag 1800 atcaaaacac tgtttctttt acggacacaa ggaaagatgg aattcacact ttacaacggt 1860 gagaagaaaa ccttctactc cagacccaac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggtatgt tgatgagcct ttctttgact gggtctacga ctcgcctgaa 1980 aacctcactc ttgaggcaat caaacagttg gaagagacaa ccggtcttga gctgcacgag 2040 ggtggaccac ccgctctcgt catctggaac atcaaacact tgcttcacac cggaatcggc 2100 actgcctcac gccctagcga ggtgtgtatg gtggacggaa cggacatgtg tttagctgat 2160 tttcatgctg gcattttcct gaaaggacag gaacatgctg tgttcgcctg tgtcacctcc 2220 aacgggtggt acgcgattga tgacgaggac ttttaccctt ggacaccgga cccgtccgac 2280 gttctggtgt ttgtcccgta cgatcaagaa ccgcttaacg gagagtggaa aacaaaggtc 2340 cagaaaaggc tcaagggagc cgggcaatcc agtccggcga ccgggtcgca gaaccagtca 2400 ggcaacactg gaagcatcat taacaactac tacatgcagc agtaccagaa ctccatggac 2460 acgcaacttg gagataacgc tatcagcgga ggctccaacg agggttccac ggacaccaca 2520 tccacacaca caaacaacac ccaaaacaat gattggttct cacgcttggc cagttcggcc 2580 tttagcggac tgtttggtgc tcttttggct gacaagaaaa cggaggagac aactctgctt 2640 gaagaccgca tactcaccac cagaaatggc cacacgacgt cgacgacaca gtcgagtgtt 2700 ggcgtaacat atggttacgc tgtggctgag gacgcggtat ctgggcctaa cacctcaggc 2760 ctggagaccc gcgtgacaca ggctgaacgg ttcttcaaga aacacctgtt tgactggacg 2820 ccggatttgt catttggaca ctgtcactac ttggaactcc cctctgaaca caagggcgtg 2880 tttggcagcc tcatgagctc ttatgcttac atgaggaacg ggtgggacat tgaggtgacc 2940 gctgttggaa atcagttcaa tggtggttgt ctcctcgtcg cactcgtgcc ggagctgaaa 3000 gagctcgaca cgcggcagaa gtatcagtta accctcttcc cacaccagtt cattaacccg 3060 cgcactaaca tgacggctca cattaacgtg ccgtacgtgg gtgtcaacag gtacgaccag 3120 tacgagctcc acaaaccgtg gacgcttgtg gtgatggtgg tggccccgct taccgtcaaa 3180 actggtggtt ctgaacagat caaggtttac atgaatgcag cgccgaccta cgtgcacgtg 3240 gcaggagaac tgccctcgaa agaggggata gttcctgtgg cgtgtgtgga cggttacggc 3300 aacatggtaa ccacggaccc gaagacggct gaccccgtct acgggaaagt gtctaacccc 3360 cccagaacaa gcttccctgg gcgcttcaca aacttccttg atgtagcgga ggcgtgtcca 3420 accttcctcc gcttcggaga agtaccattt gtgaagacgg tgaactctgg tgaccgcttg 3480 cttgccaagt ttgacgtgtc cctcgctgcg gggcacatgt ccaacaccta cttggcaggt 3540 ttggcacagt actacacaca gtacagcggc actatgaata tccacttcat gttcactgga 3600 cccacggatg ccaaagcccg ctacatggtg gcttacatac ctcctggtat gacaccgcca 3660 acggacccgg agcgggctgc acactgcatt cattctgagt gggacactgg actcaattct 3720 aaatttacct tttctatccc ttacctttct gctgcagact atgcttacac tgcttctgac 3780 gtggctgaga ccacgagtgt gcagggatgg gtgtgtattt accagatcac ccacggtaaa 3840 gctgaaggtg acgcgctggt cgtgtccgtc agcgctggca aggactttga gtttcgactg 3900 ccggtggatg cccgccaaca gactaccacc actggcgagt ccgcggaccc agtcaccacc 3960 acggttgaga actacggagg agagacccag acggcccgac ggcttcacac tgatgtcgcc 4020 ttcgttctcg acaggttcgt gaaactcacc cagcccaaga gcacccaaac ccttgatctc 4080 atgcagatcc cctcacacac actggtcggg gcgcttctcc ggtctgcgac gtactacttc 4140 tcagacctgg aggttgcgct cgtccacaca ggaccggtca cgtgggtgcc caatggtgcg 4200 cccaagaccg ccttgaacaa ccacaccaac ccgactgcct accagaagca gcctatcacc 4260 cgcttggcac tcccctacac cgctccccac cgtgtgctgt caacagtgta caacgggaag 4320 acaacgtacg gagaagaatc ctcgcggcgt ggtgatcttg ccgccctcgc aaggcgcggc 4380 gacatggcgg ccctcgcaca aaggttgcgc agagtgaaca accggctgcc cacttccttc 4440 aactacggcg ctgtgaaggc cgacaccatc acggagctgt tgatccgcat gaagcgtgcg 4500 gaaacatact gccccagacc cttgctggct cttgacacca cacaagaccg ccgtaaacag 4560 gagatcattg cacctgagaa acagcttcta aattttgacc tgctcaaatt ggcgggagat 4620 gtggagtcca accctgggcc cttcttcttc tccgacgtca ggtcaaattt ctcaaaactg 4680 gtagaaacca tcaatcagat gcaggaggac atgtcaacaa aacacgggcc tgactttaac 4740 cggttggtgt ccgcatttga ggaattggcc actggagtga aggctatcag ggccggtctc 4800 gacgaggcca aaccctggta caaactcatc aagctcctga gccgcttgtc gtgcatggcc 4860 gctgtagcag cacggtcaaa ggacccagtc cttgtggcca tcatgctggc tgacaccggt 4920 cttgagattc tggacagcac ctttgtcgtg aagaagatct ccgactcgct ctccagtctc 4980 tttcacgtgc cggcccccgt cttcagtttc ggagccccga ttctgttggc cgggttggtc 5040 aaagtcgcct cgagtttctt ccggtccaca cccgaagacc ttgagagagc agaaaaacag 5100 ctcaaagcac gtgacattaa cgacatattc gccattctca agaacggcga gtggctggtc 5160 aagctgatcc ttgccatccg cgactggatc aaagcgtgga tcgcctcaga agaaaagttt 5220 gtcaccatga cggacttggt gcctggtatc cttgaaaagc agcgggatct caacgacccg 5280 agtaagtaca aggaagccaa ggagtggctc gacaacgcgc gccaggcgtg tttgaagagc 5340 gggaacgttc acattgccaa tttgtgcaaa gtggtcgccc cggcacccag caagtcgaga 5400 cccgaacccg tggtcgtttg cctccgcggc aaatccggcc aggggaagag tttccttgcg 5460 aacgtgctcg cgcaagcaat ctccacccac ttcaccggca gaactgattc ggtttggtac 5520 tgcccgcctg accctgacca cttcgacggt tacaaccagc agaccgttgt cgtgatggac 5580 gatttgggcc agaaccccga tggcaaggac ttcaagtact tcgcccagat ggtttcgacc 5640 acggggttca tcccgcccat ggcctcgctt gaggacaaag gcaagccttt caacagcaaa 5700 gtcatcattg ctaccaccaa cctgtactcg ggtttcaccc cgagaacaat ggtgtgtcct 5760 gcgcgctga accggaggtt ccactttgac atcgacgtga gtgccaagga cgggtacaaa 5820 gttaacaaca aattggacat aatcaaagct cttgaagaca cccacaccaa cccagtggcg 5880 atgttccaat acgactgtgc ccttctaaac ggtatggcag ttgaaatgaa gagaatgcaa 5940 caggatatgt tcaagcctca accacccctc cagaacgtgt accaactcgt tcacgaggtg 6000 attgaacggg tcgagctcca cgagaaggtg tcgagccacc cgattttcaa acagatatca 6060 attccttccc aaaagtctgt gttgtacttc ctcattgaga aaggccaaca cgaagcagca 6120 attgaattct ttgagggaat ggtgcatgac tccatcaagg aagagctccg gcccctcatc 6180 caacagacct catttgtgaa acgcgctttt aagcgcctga aggaaaactt tgagactgtt 6240 gccctgtgtt tgactctttt ggcaaacata gtgatcatga tccgcgagac tcgcaagaga 6300 caacagatgg tggacgatgc agtgaatgac tacattgaga aggcaaacat caccacagat 6360 gacaagactc ttgacgaggc ggaaaagaac cctctagaga ccagcggtgc cagcactatt 6420 ggtttcagag agagaactct cccggggcac aaggcgagcg atgacgtgag ctccgagccc 6480 gccaaacccg tggaggaccg accacaagct gaagggccct acgccggacc acttaagaaa 6540 ccagtggcat tgaaagtgag agcaagagcc ccggtcgtga aggagggacc ctacgaggga 6600 ccggtgaaga agcctgtcgc tttgaaagtg aaagccaaga acttgattgt cactgagagt 6660 ggtgccccac cgaccgactt gcagaagatg gtcatgggca acactaagcc tgttgagctc 6720 atcctcgacg ggaagacggt agccatctgc tgtgctaccg gagtgtttgg cactgcctac 6780 ctcgtacctc gtcacctctt cgcggagaag tacgacaaga taatgttgga cggtagagcc 6840 atgacagaca gtgactacag agtgtttgag tttgagatta aagtaaaagg acaggacatg 6900 ctctcagacg ctgcactcat ggtgcttcac cgtgggaacc gcgtgagaga catcacgaaa 6960 cattttcgtg acacagcaag aatgaagaaa ggcacccccg ttgtcggtgt gatcaacaac 7020 gccgacgttg ggagactgat tttctctgga gaggccctta cctacaaaga cattgtagtg 7080 tgcatggatg gagacaccat gccgggcctg tttgcctaca gagccgccac caaggctggt 7140 tactgcgggg gagccgttct cgccaaggac ggagccgaca cattcatcgt tggcacccac 7200 tccgcaggtg gtaacggagt tggatactgc tcgtgcgtgt ccaggtccat gctcctgaaa 7260 atgaaggcac acattgaccc tgaaccacac cacgaggggt tgattgttga taccagagat 7320 gtggaagagc gcgtgcatgt catgcgtaaa accaagcttg cacccaccgt ggcacacggt 7380 gtgtttaacc ctgaatttgg tcccgctgcc ttgtccaaca aggacccgcg gctgaacgaa 7440 ggggttgtcc tcgatgaagt catcttctcc aaacacaagg gagacacgaa aatgtctgag 7500 gaggacaaag cgctgttccg ccgctgcgct gccgactacg cgtcgcactt gcacagcgtg 7560 ctggggacgg caaatgcccc attgagcatc tatgaggcca tcaaaggcgt cgacgggctc 7620 gatgccatgg agccggacac cgcgcccggc ctcccctggg ccctccaggg gaaacgccgt 7680 ggtgcgttga ttgacttcga gaacggcacg gtcggacccg aagtcgaggc tgccctaaag 7740 ctcatggaga aaagagagta caaatttgct tgccagacct tcctgaaaga cgagattcgt 7800 cggatggaaa aagtacgtgc tggcaagact cgcattgtcg acgttttgcc cgtggaacac 7860 attctttaca ccaggatgat gattggcaga ttctgtgctc aaatgcacac aaacaatgga 7920 ccgcagattg gctcagcggt cggttgcaat cctgatgttg attggcaaag atttggcaca 7980 cattttgctc agtacagaaa cgtgtgggat gtggactatt cggcctttga tgctaaccac 8040 tgcagtgacg caatgaacat catgtttgag gaggtatttc gcacagactt cggtttccac 8100 ccaaatgctg agtggattct gaagactctt gtgaacacgg agcacgccta tgagaacaaa 8160 cgtatcactg ttgagggcgg gatgccgtct ggctgttccg cgacaagcat catcaacaca 8220 attttgaaca acatttatgt gctctacgct cttcgtagac actatgaggg agttgagctg 8280 gacacctaca ccatgatctc ctacggagat gacatcgtgg ttgcaagtga ctacgatctg 8340 gattttgagg ctctcaaacc ccacttcaaa tctcttggtc aaaccatcac tccagctgac 8400 aaaagcgaca aaggttttgt tcttggtcac tccattaccg atgtcacttt cctcaaaaga 8460 cacttccaca tggactatgg aactgggttt tacaaacctg tgatggcctc aaagaccctc 8520 gaggccattc tctcctttgc acgccgtggg accatacagg agaagttgat ctccgtggca 8580 ggactcgccg tccactcagg acctgacgag taccggcgtc tctttgagcc cttccagggt 8640 ctcttcgaga ttccaagcta cagatcactt tacctgcgtt gggtgaacgc cgtgtgcggt 8700 gacgcataat ccctcagatg tcacaattgg cagaaagact ctgaggcgag cgacgccgta 8760 ggagtgaaaa gcccgaaagg gcttttcccg cttcctattc caaaaaaaaa aaaaaaaaac 8820 tagttctaga gcggccgcca ccgcggtgga gctccagctt ttgttccctt tagtgagggt 8880 taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 8940 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 9000 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 9060 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 9120 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 9180 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 9240 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 9300 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 9360 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 9420 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 9480 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 9540 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 9600 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 9660 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 9720 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 9780 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 9840 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 9900 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 9960 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 10020 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 10080 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 10140 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 10200 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 10260 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 10320 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 10380 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 10440 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 10500 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 10560 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 10620 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 10680 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 10740 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 10800 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 10860 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 10920 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 10980 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 11040 cccgaaaagt gccac 11055

Claims (20)

P1 gene of foot-and-mouth disease type O virus is substituted with P1 gene of Asia1 / MOG / 2005,
The VP1 site of the substituted P1 gene is substituted with the VP1 gene of As1 / Shamir / 89 shown in SEQ ID NO: 11 or the base sequence encoding the 140-160th amino acid sequence of As1 / Shamir / 89 VP1 protein at the VP1 site Lt; / RTI &gt;
Wherein the foot-and-mouth disease type O virus comprises 3B1 sites corresponding to 22 to 36 bp in SEQ ID NO: 1 and 3B2 sites corresponding to 37 to 105 bp in SEQ ID NO: 2, A recombinant vector comprising the gene of the virus. delete The recombinant vector according to claim 1, wherein the nucleotide sequence encoding the 140-160 amino acid sequence of the As1 / Shamir / 89 VP1 protein is a nucleotide sequence encoding an epitope. delete The recombinant vector according to claim 1, wherein the foot-and-mouth disease type O virus is a foot-and-mouth type O type vaccine strain Manisa. delete delete delete 2. The recombinant vector according to claim 1, wherein said recombinant vector is represented by a cleavage map of Fig.
[Figure 3]

2. The recombinant vector according to claim 1, wherein said recombinant vector is represented by a cleavage map of Fig.
[Figure 4]

2. The recombinant vector according to claim 1, wherein the recombinant vector is represented by the nucleotide sequence of SEQ ID NO: 14 or SEQ ID NO: A recombinant foot-and-mouth disease virus produced by the recombinant vector of any one of claims 1, 3, 5, 9 to 11. 1) inserting the gene of foot-and-mouth disease type O virus into a recombinant vector;
2) replacing the nucleotide sequence shown in SEQ ID NO: 1 in the 3B region of the foot-and-mouth disease type O virus in the recombination vector of the above 1) and deleting the nucleotide sequence shown in SEQ ID NO: 2;
3) replacing the P1 gene of foot-and-mouth disease type O virus with the P1 gene of Asia1 / MOG / 2005 in the recombinant vector of step 2); And
4) Replace the VP1 site of Asia1 / MOG / 2005 P1 gene of step 3) with the VP1 gene of Asia / Shamir / 89 of SEQ ID NO: 11 or substitute the VP1 gene of Asi / Shamir / Inserting a nucleotide sequence encoding an amino acid sequence; &Lt; / RTI &gt;
14. A method for producing recombinant foot-and-mouth virus comprising the step of introducing the recombinant vector prepared in claim 13 into cells. 15. The method of claim 14, wherein the cell is selected from the group consisting of a canine, a feline, a wild boar and an animal, a bovine animal, a deer and an animal, a giraffe and an animal, a fakari animal, a camel animal, a hippopotamus, Wherein the virus is derived from at least one cell selected from the group consisting of a mouse, a rabbit, a rodent, and a cell of a primate. 15. The method of claim 14, wherein the cells are selected from the group consisting of goat tongue cells (ZZ-R) and hamster kidney cells (BHK-21), black goat kidney cells (BGK), porcine kidney cells (IBRS- ). &Lt; / RTI &gt; 12. A vaccine composition for preventing foot-and-mouth disease comprising the recombinant foot-and-mouth disease virus of claim 12 as an active ingredient. 18. The vaccine composition according to claim 17, wherein the vaccine is a live vaccine, an attenuated vaccine, or a vaccine. 12. A method for preventing foot-and-mouth disease comprising administering the recombinant foot-and-mouth disease virus of claim 12 to a subject other than a human. 20. The method of claim 19, wherein the object is at least one selected from the group consisting of wild boar and animal, bovine animal, deer and animal, giraffe and animal, fecal and animal, camel, hippopotamus, rhino and animal. Prevention method.

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