KR101891607B1 - Recombinant foot-and-mouth disease virus expressing stable and differential protective antigen of Asian isolates and standard strains - Google Patents

Abstract

The present invention relates to a recombinant foot-and-mouth disease virus capable of expressing protective antigens of Asian standard strain Asia1 Shamir and foot-and-mouth disease O-type Asia strain (Asia1 MOG/2005). The present invention further relates to a production method thereof. According to the present invention, it is possible to produce a vaccine in a safe and convenient way since stability and distinction from wild types are secured through manipulation of environmental resistance factors and non-structural proteins while maximizing protective effects in wider range for foot-and-mouth disease O-type serotypes.

Description

{Recombinant foot-and-mouth disease virus expressing stable and differential protective antigens of Asian isolates and standard strains}

The present invention relates to a recombinant virus which simultaneously expresses the defensive antigen of the foot-and-mouth disease type Asia 1 type Asian vaccine and the vaccine standard state Shamir type, and a method for producing the recombinant virus.

Foot and mouth disease (FMD) is a viral vesicular disease that infects animals with split hoofs and is characterized by rapid replication and rapid spreading. Due to its economic importance, this disease is classified as a very important animal disease by the World Animal Health Organization (OIE) and it is the most important factor in the trade of livestock products because it is impossible to trade between countries at the time of occurrence.

The foot-and-mouth disease pathogen is a single-stranded bipolar RNA virus that belongs to the genus Piconaviridae and Aphthovirus and is classified into seven different serotypes (A, O, C, Asia1, SAT1, SAT2 and SAT3) .

Foot-and-mouth disease virus There are six genotypes (Genetic lineage I to VI) among Asia1 types. This type of vaccine is required to develop various types of vaccines against Asia 1 type virus because it is not protected against virus attack because of high rate of change even in this region type.

However, in order to develop all the vaccine strains, the outdoors must be continuously cultured in the cells to have an adequate propagation capacity. However, this method is time consuming and there is no guarantee that the results will be good. Therefore, it should be made in a form that can be produced in a timely manner so that new vaccine strains can be quickly developed and used by using a lot of existing information and already proven vaccine viruses.

In order to solve the above problem, the inventors of the present invention have found that, by using a basic vector composed of a viral gene based on a pathogenic weak virus, which is already known as a foot-and-mouth disease vaccine, A new recombinant virus capable of producing a protective vaccine was prepared.

Korean Patent No. 10-1566371

Accordingly, it is an object of the present invention to provide a recombinant foot-and-mouth disease virus which can be used as a vaccine seedlable by simultaneously providing defense against foot-and-mouth disease and vaccine shamir.

Another object of the present invention is to provide a method for producing the recombinant virus.

It is still another object of the present invention 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 inventors have conducted studies to develop a stable vaccine strain having a broader range of defense against Asia 1 type of foot-and-mouth disease virus, and found that a gene coding for a structural part of the Asia 1 type virus and a non- The present inventors have found that a recombinant virus that has been manipulated at a specific site of the present invention can be distinguished from an open field with environmental resistance and can produce a stable vaccine.

That is, according to the present invention, the following invention is provided.

1) The recombinant plasmid containing the gene of foot-and-mouth disease O virus is obtained by replacing P1 gene of foot-and-mouth disease type O virus with P1 gene of Asia1 MOG, and VP1 of the substituted P1 gene is substituted with VP1 gene of Asia1 Shamir, (H145Y) of the 145th amino acid histidine at the VP2 site among the P1 genes of the substituted Asia1 MOG, and the 17th amino acid asparagine of the substituted VP1 region of Asia1 Sham is substituted (N17D) with aspartic acid, A recombinant plasmid comprising the gene of foot-and-mouth disease type O virus, wherein the foot-and-mouth disease type O virus is substituted in 3B12 site among 3B sites.

2) Recombinant foot-and mouth disease virus produced using the recombinant plasmid.

3) The recombinant foot-and-mouth disease virus, wherein the protective antigen of Asia 1 MOG and Asia 1 Shamir is expressed simultaneously.

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

(b) replacing the P1 gene of foot-and-mouth disease type O virus with the P1 gene of Asia 1 MOG in the recombinant vector of step (a);

(c) replacing the VP1 site of the P1 gene of Asia1 MOG with the VP1 gene of Asia1 Shamir in the step (b);

(d) substituting tyrosine (H145Y) for the 145th amino acid histidine at the VP2 site of the P1 gene of the substituted Asia1 MOG in the recombinant vector of step (c), and isolating the 17th amino acid asparagine (N17D) with an aspartic acid;

(e) replacing the 3B12 region of foot-and-mouth disease type O virus in the recombinant vector of step (d).

5) introducing the recombinant vector prepared above into a cell and propagating the recombinant vector.

6) A vaccine composition for preventing foot-and-mouth disease comprising the recombinant FMDV as an active ingredient.

7) A method for preventing foot-and-mouth disease comprising administering the recombinant foot-and-mouth virus to an individual other than a human.

The recombinant foot-and-mouth disease virus according to the present invention has an advantage that it can be used more safely than a pathogenic virus because it is treated in a laboratory such as a disinfectant experiment or an antiviral experiment by weakening the pathogenicity of an animal by manipulating a part of a gene of foot-

In addition, by producing the vaccine using the recombinant foot-and-mouth disease virus according to the present invention as an active ingredient, the virus titer can be substantially prevented from being different from the original virus, There is an effect.

In addition, the recombinant foot-and-mouth disease virus according to the present invention is manufactured to induce the environmental resistance of the antigen and is resistant and can be used for vaccine production because it can be diagnosed differently from the open field.

1 shows a genomic diagram of a recombinant FMDV virus expressing a stable protective antigen in the foot-and-mouth disease Asia 1 serotype Asia (Mongolia) origin and standard.
Fig. 2 is a graph showing the results of a comparison between plasmids in which two environmental resistance factors (one VP1 and one VP2 internal gene) in the plasmid of Fig. 1 are replaced, 3B1 and 3B2 sites of foot-and-mouth disease O Manisa are replaced with 3B3 of A- Fig.
FIG. 3 shows that the expression of an antigen in the infected cells of the rAsiaM-Sham-3B foot-and-mouth disease virus was confirmed by a simple antigen kit (band formation of SP band and non-formation of NSP) Serologically, it is also possible to distinguish it from open field.
FIG. 4 shows that the rAsiaM-Sham-3B foot-and-mouth disease virus with the replaced gene as shown in FIG. 2 showed resistance by treatment with pH 6.0, and that the virus activity was not inhibited at 42 hours at 42.degree.
FIG. 5 shows survival rate and weight loss of mice through an attack inoculation experiment (Asia 1 Shamir) after 7 days immunization (0.25-0.125 μg, 1 / 60-1 / 120 dose) in mice. , It was confirmed that the antigen of Asia1 Shamir virus was no problem in defense of the virus.
FIG. 6 is a graph showing the results of immunization experiment (Asia 1 Shamir) in which mice were immunized for 7 days after antigen immunization and diluted more (0.25-0.125 μg, 1 / 10-1 / 320 dose) And the survival rate of mice. According to these results, it was confirmed that even inoculated with a vaccine diluted to 0.046 μg (1/320 dose), it is possible to protect against Asia 1 Shamir virus.
FIG. 7 shows the results of immunization (1.5 μg, 1/10 dose) after inoculation of the tester vaccine in mice for 2 months at 4 ° C. to measure the stability (stability) As a result, all the mice survived and were found to be conserved.
FIG. 8 shows the results of immunological correlations between ELISA antibody level and each virus for sera immunized with the sera obtained through immunization test (guinea pig, two weeks after the first vaccination and one week after the second vaccination). Asia1 MOG showed neutralizing antibody formation almost similar to that of vaccine strain. Asia1 Shamir and Asia1 LC04 showed lower antibody formation.
FIG. 9 shows the results of an immunological correlation test conducted through an immunological experiment in cattle (blood sampling 3 weeks after the first inoculation). Similar to the case of guinea pigs, Asian1 MOG showed almost the same neutralizing antibody formation as the vaccine strain. Asia1 Shamir was the next best, and Asia1 LC04 showed lower antibody formation.
Fig. 10 shows the antibody levels confirmed by immunological experiments in pigs by ELISA and neutralization test. ELISA and neutralizing antibodies of the vaccine antibody were better formed than the control trivalent vaccine (O Mansia, A22 Iraq, Asia 1 Shamir type trivalent vaccine, 6PD50 or higher).

The present invention relates to a recombinant plasmid having the nucleotide sequence of SEQ ID NO: 1, which comprises a gene of foot-and-mouth disease type O virus, wherein P1 gene of foot-and-mouth disease type O virus is substituted with P1 gene of Asia1 MOG, The VP1 site is substituted with the VP1 gene of Asia1 Shamir, and the 145th amino acid histidine at the VP2 site among the P1 genes of the substituted Asia1 MOG is replaced with tyrosine (H145Y), and the 17th amino acid Wherein the asparagine is replaced with aspartic acid (N17D), and the foot-and-mouth disease O virus is replaced with a 3B12 site in the 3B region.

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 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. The 3B1 region and the 3B2 region may be partially substituted in the entire gene region. The substitution sites of 3B1 and 3B2 are preferably 6474 to 6617 bp in the recombinant plasmid of the present invention represented by the nucleotide sequence of SEQ ID NO: 1, but are 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.

According to another embodiment of the present invention, the present invention relates to a recombinant foot-and-mouth virus produced using the recombinant plasmid.

The recombinant virus of the present invention is characterized in that the protective antigen of foot-and-mouth disease Asia 1 MOG and Asia 1 Shamir are simultaneously expressed.

According to another embodiment of the present invention,

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

(b) replacing the P1 gene of foot-and-mouth disease type O virus with the P1 gene of Asia 1 MOG in the recombinant vector of step (a);

(c) replacing the VP1 site of the P1 gene of Asia1 MOG with the VP1 gene of Asia1 Shamir in the step (b);

(d) substituting tyrosine (H145Y) for the 145th amino acid histidine at the VP2 site of the P1 gene of the substituted Asia1 MOG in the recombinant vector of step (c), and isolating the 17th amino acid asparagine (N17D) with an aspartic acid;

(d) replacing the 3B12 region of foot-and-mouth disease type O virus in the recombinant vector of step (d).

According to another embodiment of the present invention, the present invention relates to a method for producing recombinant foot-and-mouth virus including the step of introducing a recombinant vector produced by a production method into a cell to proliferate.

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.

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, (ZZR) and hamster kidney cells (BHK-21), black goat kidney cells (BGK), pig kidney cells (BGK), and the like. (IBRS-2) and small intestine cells (LF-BK).

According to another embodiment of the present invention, the present invention relates to a vaccine composition for preventing foot-and-mouth disease comprising the recombinant FMDV as an active ingredient.

In the foot-and-mouth disease vaccine composition of the present invention, the vaccine may be a live vaccine, an attenuated vaccine, or a vaccine.

The vaccine composition of the present invention may contain one or more known active ingredients having excellent preventive effect against foot-and-mouth disease with recombinant foot-and-mouth disease virus.

The vaccine 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.

The vaccine composition of the present invention may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dose may vary depending on the weight, age, The range varies depending on the condition, diet, administration time, method of administration, excretion rate, and severity of the disease.

According to another embodiment of the present invention, the present invention relates to a method for preventing foot-and-mouth disease comprising administering the recombinant foot-and-mouth disease virus to a subject other than a human.

Hereinafter, the content 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. Environment  Production of Resistant Plasmids

The recombinant plasmid pO-Manisa was constructed by amplifying the entire O-Manisa virus gene (GenBank Accession No. AY593823.1) by PCR and inserting the O-Manisa gene into a plasmid (pBluescript SK II).

Among the recombinant plasmids (pO-Manisa) prepared above, the gene coding for the P1 protein as a defense antigen was amplified by the protective antigen of Asia1 / MOG / 2005 virus (GenBank Accession No. EF614458.1) (POm-AsMOG-P1) into which a recombinant gene was inserted.

Based on the plasmid (pOm-AsMOG-P1) prepared above, the primer for VP1 amplification of the vaccine standard host As1 / Shamir / 89 was prepared using the forward primer (5'-ACCACCACCACTGGCGAATCAGCAGAT-3 ') of SEQ ID NO: (5'-CTTCTAAATTTTGACCTGCTCAAATTGGCGGG-3 ') of SEQ ID NO: 9 and the reverse primer (5'-CTGTTGGCGGGCATCCACCGGCAGTCG-3') of SEQ ID NO: 10 were used to amplify the vector, and the reverse primer (5'-CTGCTTCTCAGGTGCAATGATCTCC- '), The pOm-AsM-ShamVP1 plasmid (Patent No. 10-1566371, Oct. 30, 2015) was prepared by ligating the VP1 gene of As1 / Shamir / 89 with pOm-AsMOG-P1.

An environmentally resistant plasmid was prepared using the pOm-Asm-ShamVP1 plasmid. Asm VP2 To replace the H145Y site, Asm VP2 H145Y F (5'-TCCCATACCAGTTCATTAACCCGC-3; SEQ ID NO: 2) and Sham VP1 N17D R5'-CTGAGTCTCTCCTCCATAGTCCTC-3; (SEQ ID NO: 3) primer, and the forward primer (5'-ACAGCCAGACGGCTTCACACTGAC-3 ') of SEQ ID NO: 4 and the reverse primer (5'-AGAGGGTTAACTGATACTTCTGCC-3' After amplification, the replaced recombinant plasmid was ligated (TAKARA Long Ligation kit) and finally cloned (pOm-Asm VP2 H145Y sham VP1 N17D ).

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), 10 mM dNTPs (1 占 퐇), Phusion enzyme (2 U / 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. After that, a ligated reaction with the amplified P1 gene (TAKARA Long Ligation kit) was performed. Finally, it was confirmed that cloning was properly performed through the whole base sequence analysis.

Plasmid primer name Primer base sequence SEQ ID NO:
rAsia - MOG - Sham -3B O1m ASM VP2 H145Y F 5'-TCCCATACCAGTTCATTAACCCGC-3 ' SEQ ID NO: 2 O1m Sham VP1 N17D R 5'-CTGAGTCTCTCCTCCATAGTCCTC-3 ' SEQ ID NO: 3 vector F 5'-ACAGCCAGACGGCTTCACACTGAC -3 ' SEQ ID NO: 4 vector R 5'-AGAGGGTTAACTGATACTTCTGCC -3 ' SEQ ID NO: 5

Example  2. Foot and mouth disease Outdoor week  For differential diagnosis rAsiaM - Sham Production of -3B

The 3B region (3B333) of the environmentally resistant plasmid in which the Asm VP2 H145Y region and the Sham VP1 N17D region were replaced in Example 1 was synthesized from the synthesized sequence (144 bps, corresponding to positions 6474 to 6617 bp in SEQ ID NO: 1) 3B3 of A type and 3B3 of SAT type were synthesized and inserted.

Generally, cDNA of O-Manisa virus is generated using a random primer used in cDNA synthesis. Based on the cloning of the whole gene, O-Manisa virus based on the gene information, the end portion gene of 3A and the start region gene of 3B3 PCR was performed by preparing specific primers (corresponding to 20 mers of 5 'and 3' genes on the basis of GenBank Accession No. AY593823.1). Conditions for PCR were 5X buffer (FINNZYMES, 10 μl ), 10 mM dNTPs (1 μl), Phusion enzyme (2 U / μl, 0.5 μl) and sterilized distilled water (35.5 μl) at 98 ° C. for 30 seconds, 98 ° C. for 10 seconds, 65 ° C. for 30 seconds, and 72 ° C. for 2 minutes and 30 seconds 25 cycles, and final 72 ° C for 10 minutes. (TAKARA Long Ligation kit) was performed and the cloning was confirmed through the whole sequence analysis. Table 2 below shows amino acid sequences in which 3B1 and 3B2 were removed and 3B3 of type A and 3B3 of SAT type were inserted.

NSP site manipulation Amino acid sequence Amino acid size Reference 3B12
deletion GPYAGPLERQKPLRVKTKLPQQEGPYAGPMDRQKPLKVRARAPVVKE 47 aa - 3B33
addition GPYEGPVKKPVALKVKAKNLIVTEGPYEGPVKKPVALKVKAKAPIVTE 48 aa A type and SAT type

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

Recovered recombinant foot-and-mouth disease virus was recovered by reacting the obtained recombinant plasmid (rAsiaM-Sham-3B) with a restriction enzyme to prepare a single fragment of the gene, and the BHKT7-9 cell (a cell line expressing T7 RNA polymerase) The purified DNA was transformed using a transformation reagent (Life Technologies) and cultured for 2 to 3 days to obtain a recombinant virus. Thereafter, the virus was propagated by inoculating the obtained virus through a continuous passage in ZZ-R (fetal tongue) cells or BHK-21 (young hamster kidney) cells.

Example  4. Identification of the resistance of the production virus and immunogenicity in animals

The virus prepared as described above was used as a control for viruses in which the VP1 and VP2 genes were not manipulated. To confirm the stability of the virus prepared as a stable virus, pH 6.0 was treated and the virus was treated at 42 ° C for 2 hours The virus titers were then checked to compare the virus titers. As a result of comparison, virus resistance was low in general foot - and - mouth disease virus because it was sensitive to pH.

For animal experiments, 0.5 ml of purified foot-and-mouth disease virus antigen (15 μg / dose) was mixed with 0.5 ml of adjuvant containing oil adjuvant ISA206 to prepare double oil. The mice were treated with 0.1 ml of mouse, 0.2 ml of guinea pig, and pigs and cows using mouse (C57 / BL6), guinea pigs, 10-12 week old pigs, 1ml, and mice were immunized for 7 ~ 21 days after each immunization. Body weight change and survival rate were observed. Guinea pigs, pigs and cows were bled for each antibody and Asia1 SP-ELISA (OIE manual standard test method) was performed by using a kit (self-contained kit of Prionic kit) and a vaccine strain to determine the antibody level.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventive concept as defined by the appended claims. It will be understood that various modifications and changes may be made in the present invention.

By providing a foot-and-mouth disease vaccine using the recombinant foot-and-mouth disease virus according to the present invention having broad-spectrum antigen-expressing ability of foot-and-mouth disease Asia 1 serotype, it can contribute to the treatment and prevention of foot-and-mouth disease.

<110> Animal and plant quaratine agency <120> Recombinant foot-and-mouth disease virus expressing stable and          differential protective antigen of Asian isolates and standard          strains <130> 17-10709 <160> 5 <170> KoPatentin 3.0 <210> 1 <211> 11087 <212> DNA <213> Artificial Sequence <220> The rAsiaM-Sham-3B plasmid <400> 1 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 ctaggtttta ccgtcgttcc cgacgttaat ggggaaacaa ccacaagctt 1080 aacaccgtct tgcccgacgt aaaagggctg caaccaaaaa gcttgtgccg cctttcccgg 1140 cgttaatggg aggtaaccac aagacaaacc ttcacccgga agtaaaacgg caacttcaca 1200 cagttttgcc cgttttcgtg agaaatgggc cgtcaacgca cgaaacgcgc cgtcgcttga 1260 ggaggacttg tacaaacacg atctatgcag gtttccacaa ctgacacaaa ccgtgcaact 1320 tgaaaccccg cctggtcttt ccaggtctag aggggcgaca ttttgtactg tgcttgactc 1380 cacgctcggt ccactagcga gtgttagtag tagcactgtt gcttcgtagc ggagcatgat 1440 ggccgtggga gcttcccctt ggtaacaagg acccacgggg ccaaaagcca cgtcctaccg 1500 gacccatcat gtgtgcaaac ccagcacggc aactttactg cgaaaaccac tttaaggtga 1560 cactgatact ggtactcaat cactggtgac aggctaagga tgcccttcag gtaccccgag 1620 gtaacacgcg acactcggga tctgagaagg ggactggggc ttctttaaaa gtgcccagtt 1680 taaaaagctt ctatgcctga ataggcgacc ggaggccggc gccttttcac tgttttacta 1740 ctgttttcat gaatacaact gactgtttca ccgccctgtt acacgctctc agagagatca 1800 aaacactgtt tcttttacgg acacaaggaa agatggaatt cacactttac aacggtgaga 1860 agaaaacctt ctactccaga cccaacaacc acgacaactg ctggcttaac accattctcc 1920 agttgttcag gtatgttgat gagcctttct ttgactgggt ctacgactcg cctgaaaacc 1980 tcactcttga ggcaatcaaa cagttggaag agacaaccgg tcttgagctg cacgagggtg 2040 gaccacccgc tctcgtcatc tggaacatca aacacttgct tcacaccgga atcggcactg 2100 cctcacgccc tagcgaggtg tgtatggtgg acggaacgga catgtgttta gctgattttc 2160 atgctggcat tttcctgaaa ggacaggaac atgctgtgtt cgcctgtgtc acctccaacg 2220 ggtggtacgc gattgatgac gaggactttt acccttggac accggacccg tccgacgttc 2280 tggtgtttgt cccgtacgat caagaaccgc ttaacggaga gtggaaaaca aaggtccaga 2340 aaaggctcaa gggagccggg caatccagtc cggcgaccgg gtcgcagaac cagtcaggca 2400 acactggaag catcattaac aactactaca tgcagcagta ccagaactcc atggacacgc 2460 aacttggaga taacgctatc agcggaggct ccaacgaggg ttccacggac accacatcca 2520 cacacacaaa caacacccaa aacaatgatt ggttctcacg cttggccagt tcggccttta 2580 gcggactgtt tggtgctctt ttggctgaca agaaaacgga ggagacaact ctgcttgaag 2640 accgcatact caccaccaga aatggccaca cgacgtcgac gacacagtcg agtgttggcg 2700 taacatatgg ttacgctgtg gctgaggacg cggtatctgg gcctaacacc tcaggcctgg 2760 agacccgcgt gacacaggct gaacggttct tcaagaaaca cctgtttgac tggacgccgg 2820 atttgtcatt tggacactgt cactacttgg aactcccctc tgaacacaag ggcgtgtttg 2880 gcagcctcat gagctcttat gcttacatga ggaacgggtg ggacattgag gtgaccgctg 2940 ttggaaatca gttcaatggt ggttgtctcc tcgtcgcact cgtgccggag ctgaaagagc 3000 tcgacacgcg gcagaagtat cagttaaccc tcttcccata ccagttcatt aacccgcgca 3060 ctaacatgac ggctcacatt aacgtgccgt acgtgggtgt caacaggtac gaccagtacg 3120 agctccacaa accgtggacg cttgtggtga tggtggtggc cccgcttacc gtcaaaactg 3180 gtggttctga acagatcaag gtttacatga atgcagcgcc gacctacgtg cacgtggcag 3240 gagaactgcc ctcgaaagag gggatagttc ctgtggcgtg tgtggacggt tacggcaaca 3300 tggtaaccac ggacccgaag acggctgacc ccgtctacgg gaaagtgtct aaccccccca 3360 gaacaagctt ccctgggcgc ttcacaaact tccttgatgt agcggaggcg tgtccaacct 3420 tcctccgctt cggagaagta ccatttgtga agacggtgaa ctctggtgac cgcttgcttg 3480 ccaagtttga cgtgtccctc gctgcggggc acatgtccaa cacctacttg gcaggtttgg 3540 cacagtacta cacacagtac agcggcacta tgaatatcca cttcatgttc actggaccca 3600 cggatgccaa agcccgctac atggtggctt acatacctcc tggtatgaca ccgccaacgg 3660 acccggagcg ggctgcacac tgcattcatt ctgagtggga cactggactc aattctaaat 3720 ttaccttttc tatcccttac ctttctgctg cagactatgc ttacactgct tctgacgcgg 3780 ctgagaccac gagtgtgcag ggatgggtgt gtatttacca gatcacccac ggtaaagctg 3840 aaggtgacgc gctggtcgtg tccgtcagcg ctggcaagga ctttgagttt cgactgccgg 3900 tggatgcccg ccaacagact accaccactg gcgaatcagc agatccagtc acaaccacgg 3960 ttgaggacta tggaggagag actcagacag ccagacggct tcacactgac gtcgccttca 4020 ttcttgacag gtttgtgaaa ctcactgctc ccaagaacat ccaaaccctc gatctcatgc 4080 agatcccctc acacacgctg gttggagcac tacttcgttc tgcgacgtac tacttctcag 4140 acctggaggt cgcgcttgtc cacacaggcc cggtcacctg ggtgcccaac ggcgcgccca 4200 aggatgctct aaacaaccag accaacccaa ctgcctatca gaagcaaccc atcacccgcc 4260 tggcactccc ctacaccgcc ccccatcgtg tgctggcaac agtgtacaac gggaagacgg 4320 cgtacgggga aacgacctca aggcgcggcg acatggcggc cctcgcacaa aggttgagcg 4380 ctcggctgcc cacctccttc aactacggcg ccgtgaaggc cgacaccatc actgagcttt 4440 tgatccgcat gaagcgcgcg gagacatatt gccctaggcc tttactagcc cttgacacca 4500 ctcaggaccg ccgcaaacag gagatcattg cacctgagaa gcagcttcta aattttgacc 4560 tgctcaaatt ggcgggagat gtggagtcca accctgggcc cttcttcttc tccgacgtca 4620 ggtcaaattt ctcaaaactg gtagaaacca tcaatcagat gcaggaggac atgtcaacaa 4680 aacacgggcc tgactttaac cggttggtgt ccgcatttga ggaattggcc actggagtga 4740 aggctatcag ggccggtctc gacgaggcca aaccctggta caaactcatc aagctcctga 4800 gccgcttgtc gtgcatggcc gctgtagcag cacggtcaaa ggacccagtc cttgtggcca 4860 tcatgctggc tgacaccggt cttgagattc tggacagcac ctttgtcgtg aagaagatct 4920 ccgactcgct ctccagtctc tttcacgtgc cggcccccgt cttcagtttc ggagccccga 4980 ttctgttggc cgggttggtc aaagtcgcct cgagtttctt ccggtccaca cccgaagacc 5040 ttgagagagc agaaaaacag ctcaaagcac gtgacattaa cgacatattc gccattctca 5100 agaacggcga gtggctggtc aagctgatcc ttgccatccg cgactggatc aaagcgtgga 5160 tcgcctcaga agaaaagttt gtcaccatga cggacttggt gcctggtatc cttgaaaagc 5220 agcgggatct caacgacccg agtaagtaca aggaagccaa ggagtggctc gacaacgcgc 5280 gccaggcgtg tttgaagagc gggaacgttc acattgccaa tttgtgcaaa gtggtcgccc 5340 cggcacccag caagtcgaga cccgaacccg tggtcgtttg cctccgcggc aaatccggcc 5400 aggggaagag tttccttgcg aacgtgctcg cgcaagcaat ctccacccac ttcaccggca 5460 gaactgattc ggtttggtac tgcccgcctg accctgacca cttcgacggt tacaaccagc 5520 agaccgttgt cgtgatggac gatttgggcc agaaccccga tggcaaggac ttcaagtact 5580 tcgcccagat ggtttcgacc acggggttca tcccgcccat ggcctcgctt gaggacaaag 5640 gcaagccttt caacagcaaa gtcatcattg ctaccaccaa cctgtactcg ggtttcaccc 5700 cgagaacaat ggtgtgtcct gacgcgctga accggaggtt ccactttgac atcgacgtga 5760 gtgccaagga cgggtacaaa gttaacaaca aattggacat aatcaaagct cttgaagaca 5820 cccacaccaa cccagtggcg atgttccaat acgactgtgc ccttctaaac ggtatggcag 5880 ttgaaatgaa gagaatgcaa caggatatgt tcaagcctca accacccctc cagaacgtgt 5940 accaactcgt tcacgaggtg attgaacggg tcgagctcca cgagaaggtg tcgagccacc 6000 cgattttcaa acagatatca attccttccc aaaagtctgt gttgtacttc ctcattgaga 6060 aaggccaaca cgaagcagca attgaattct ttgagggaat ggtgcatgac tccatcaagg 6120 aagagctccg gcccctcatc caacagacct catttgtgaa acgcgctttt aagcgcctga 6180 aggaaaactt tgagactgtt gccctgtgtt tgactctttt ggcaaacata gtgatcatga 6240 tccgcgagac tcgcaagaga caacagatgg tggacgatgc agtgaatgac tacattgaga 6300 aggcaaacat caccacagat gacaagactc ttgacgaggc ggaaaagaac cctctagaga 6360 ccagcggtgc cagcactatt ggtttcagag agagaactct cccggggcac aaggcgagcg 6420 atgacgtgag ctccgagccc gccaaacccg tggaggaccg accacaagct gaaggacctt 6480 acgagggacc ggtgaagaag cctgtcgctt tgaaagtgaa agctaagaac ttgattgtca 6540 ctgaggggcc atatgaagga ccagtgaaga aacctgtcgc tttgaaagtg aaagcaaaag 6600 ccccgattgt cactgaagga ccctacgagg gaccggtgaa gaagcctgtc gctttgaaag 6660 tgaaagccaa gaacttgatt gtcactgaga gtggtgcccc accgaccgac ttgcagaaga 6720 tggtcatggg caacactaag cctgttgagc tcatcctcga cgggaagacg gtagccatct 6780 gctgtgctac cggagtgttt ggcactgcct acctcgtacc tcgtcacctc ttcgcggaga 6840 agtacgacaa gataatgttg gacggtagag ccatgacaga cagtgactac agagtgtttg 6900 agtttgagat taaagtaaaa ggacaggaca tgctctcaga cgctgcactc atggtgcttc 6960 accgtgggaa ccgcgtgaga gacatcacga aacattttcg tgacacagca agaatgaaga 7020 aaggcacccc cgttgtcggt gtgatcaaca acgccgacgt tgggagactg attttctctg 7080 gagaggccct tacctacaaa gacattgtag tgtgcatgga tggagacacc atgccgggcc 7140 tgtttgccta cagagccgcc accaaggctg gttactgcgg gggagccgtt ctcgccaagg 7200 acggagccga cacattcatc gttggcaccc actccgcagg tggtaacgga gttggatact 7260 gctcgtgcgt gtccaggtcc atgctcctga aaatgaaggc acacattgac cctgaaccac 7320 accacgaggg gttgattgtt gataccagag atgtggaaga gcgcgtgcat gtcatgcgta 7380 aaaccaagct tgcacccacc gtggcacacg gtgtgtttaa ccctgaattt ggtcccgctg 7440 ccttgtccaa caaggacccg cggctgaacg aaggggttgt cctcgatgaa gtcatcttct 7500 ccaaacacaa gggagacacg aaaatgtctg aggaggacaa agcgctgttc cgccgctgcg 7560 ctgccgacta cgcgtcgcac ttgcacagcg tgctggggac ggcaaatgcc ccattgagca 7620 tctatgaggc catcaaaggc gtcgacgggc tcgatgccat ggagccggac accgcgcccg 7680 gcctcccctg ggccctccag gggaaacgcc gtggtgcgtt gattgacttc gagaacggca 7740 cggtcggacc cgaagtcgag gctgccctaa agctcatgga gaaaagagag tacaaatttg 7800 cttgccagac cttcctgaaa gacgagattc gtccgatgga aaaagtacgt gctggcaaga 7860 ctcgcattgt cgacgttttg cccgtggaac acattcttta caccaggatg atgattggca 7920 gattctgtgc tcaaatgcac acaaacaatg gaccgcagat tggctcagcg gtcggttgca 7980 atcctgatgt tgattggcaa agatttggca cacattttgc tcagtacaga aacgtgtggg 8040 atgtggacta ttcggccttt gatgctaacc actgcagtga cgcaatgaac atcatgtttg 8100 aggaggtatt tcgcacagac ttcggtttcc acccaaatgc tgagtggatt ctgaagactc 8160 ttgtgaacac ggagcacgcc tatgagaaca aacgtatcac tgttgagggc gggatgccgt 8220 ctggctgttc cgcgacaagc atcatcaaca caattttgaa caacatttat gtgctctacg 8280 ctcttcgtag acactatgag ggagttgagc tggacaccta caccatgatc tcctacggag 8340 atgacatcgt ggttgcaagt gactacgatc tggattttga ggctctcaaa ccccacttca 8400 aatctcttgg tcaaaccatc actccagctg acaaaagcga caaaggtttt gttcttggtc 8460 actccattac cgatgtcact ttcctcaaaa gacacttcca catggactat ggaactgggt 8520 tttacaaacc tgtgatggcc tcaaagaccc tcgaggccat tctctccttt gcacgccgtg 8580 ggaccataca ggagaagttg atctccgtgg caggactcgc cgtccactca ggacctgacg 8640 agtaccggcg tctctttgag cccttccagg gtctcttcga gattccaagc tacagatcac 8700 tttacctgcg ttgggtgaac gccgtgtgcg gtgacgcata atccctcaga tgtcacaatt 8760 ggcagaaaga ctctgaggcg agcgacgccg taggagtgaa aagcccgaaa gggcttttcc 8820 cgcttcctat tccaaaaaaa aaaaaaaaaa actagttcta gagcggccgc caccgcggtg 8880 gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc 8940 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg 9000 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt 9060 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg 9120 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga 9180 ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat 9240 acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca 9300 aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc 9360 tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata 9420 aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc 9480 gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc 9540 acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga 9600 accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc 9660 ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag 9720 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag 9780 gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag 9840 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca 9900 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga 9960 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat 10020 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga 10080 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg 10140 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga 10200 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc 10260 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac 10320 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc 10380 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc 10440 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc 10500 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt 10560 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc 10620 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg 10680 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag 10740 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat 10800 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc 10860 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa 10920 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta 10980 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa 11040 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac 11087 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for Asm VP2 <400> 2 tcccatacca gttcattaac ccgc 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for Sham VP1 <400> 3 ctgagtctct cctccatagt cctc 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rAsiaM-Sham vector <400> 4 acagccagac ggcttcacac tgac 24 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rAsiaM-Sham vector <400> 5 agagggttaa ctgatacttc tgc 23

Claims (10)

The recombinant plasmid containing the gene of foot-and-mouth disease type O virus is such that P1 gene of foot-and-mouth disease type O virus is substituted with P1 gene of Asia 1 MOG,
The VP1 site of the substituted P1 gene is substituted with the VP1 gene of Asia1 Shamir,
The 145th amino acid histidine at the VP2 site among the P1 genes of the substituted Asia1 MOG is substituted with tyrosine (H145Y), the 17th amino acid asparagine of the substituted VP1 region of Asia1 Shamir is substituted (N17D) with aspartic acid,
The foot-and-mouth disease type O virus is a recombinant plasmid comprising a gene of foot-and-mouth disease type O virus, wherein the 3B12 site in the 3B site is substituted with the 3B33 site,
Wherein the recombinant plasmid has the nucleotide sequence of SEQ ID NO: 1. delete A recombinant foot-and-mouth disease virus produced using the recombinant plasmid of claim 1. 4. The recombinant FMD virus according to claim 3, wherein said recombinant virus is expressed simultaneously in a protective antigen of Asia 1 MOG and Asia 1 Shamir. (a) inserting a gene of foot-and-mouth disease type O virus into a recombinant vector;
(b) replacing the P1 gene of foot-and-mouth disease type O virus with the P1 gene of Asia 1 MOG in the recombinant vector of step (a);
(c) replacing the VP1 site of the P1 gene of Asia1 MOG with the VP1 gene of Asia1 Shamir in the step (b);
(d) substituting tyrosine (H145Y) for the 145th amino acid histidine at the VP2 site of the P1 gene of the substituted Asia1 MOG in the recombinant vector of step (c), and isolating the 17th amino acid asparagine (N17D) with an aspartic acid;
(e) replacing the 3B12 site of the foot-and-mouth disease type O virus with the 3B33 site in the recombinant vector of step (d). A method for producing recombinant FMDV comprising the steps of: introducing the recombinant vector prepared in claim 5 into a cell to proliferate. The method of claim 6, wherein the cells are at least one selected from the group consisting of fetal goat fetal tongue (ZZ-R) and hamster kidney (BHK-21) cells. A vaccine composition for preventing foot-and-mouth disease comprising the recombinant foot-and-mouth disease virus of claim 7 as an active ingredient. The vaccine composition according to claim 8, wherein the vaccine is a live vaccine, an attenuated vaccine, or a vaccine. 4. A method for preventing foot-and-mouth disease, comprising administering the recombinant foot-and-mouth disease virus of claim 3 or 4 to an individual other than a human.

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