KR102009266B1 - Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT2 ZIM - Google Patents

Abstract

The present invention relates to a recombinant foot-and-mouth virus that expresses the protective antigen of foot-and-mouth disease SAT type II regional type ZIM and a method for producing the same, which can maximize the protective effect in a wider range for the foot-and-mouth disease SAT2 serotype.

Description

Recombinant foot-and-mouth disease virus expressing protective antigen of type SAT2 ZIM}

The present invention relates to a recombinant virus expressing the protective antigen of foot-and-mouth disease SAT2 type II regional type ZIM, a preparation method thereof, and a use as a vaccine.

Foot and mouth disease (FMD) is a viral bullous disease that infects animals with two hoofs and is characterized by rapid replication and rapid spread. This disease is classified as a very important animal disease by the World Animal Health Organization (OIE) due to its economic importance, and it is the most important factor in the trade of livestock products because it is impossible to trade between countries when it occurs.

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

Foot and mouth disease virus There are 14 regional types of SAT2 with different characteristics. If rigorously developed, all vaccines against these regional or subtypes should be developed and used as vaccine strains. However, in order to develop all vaccine strains, it is necessary to continuously culture the field strains in cells to have a proper proliferation ability. This method is time-consuming and there is no guarantee that the result will be good. Therefore, it is necessary to make a form that can be produced in a timely manner so that new vaccines can be rapidly developed and used by using existing information and proven vaccine virus.

In order to solve the above problems, the present inventors use a base vector composed of a viral gene based on a pathogenic weakened virus, which is already known as a foot-and-mouth vaccine, a novel recombinant virus that can produce a vaccine that can protect against SAT1-type foot-and-mouth disease. Efforts were continuously made to produce the Patent document 0001 discloses a recombinant plasmid in which a gene encoding a defensive antigen protein of foot-and-mouth disease SAT type 2 VII region is inserted as one of the prior studies of the present inventors, but the genetic homology of SAT2 ZIM virus shows a big difference. There is still a need for development of additional vaccine strains that can play a complementary role in these vaccine viruses. Foot-and-mouth disease is a lot of type II in addition to the SAT2 VII regional type and the present inventors confirmed that the protective effect is maximized in the form of a virus with reduced pathogenicity with a new foot-and-mouth disease recombinant virus that requires defense.

Korea Patent Registration No. 10-1602927

It is therefore an object of the present invention to provide a recombinant plasmid in which a recombinant gene encoding the II antigenic protective antigen protein of the SAT type 2 virus of foot-and-mouth disease is inserted.

Another object of the present invention is to provide a recombinant foot-and-mouth virus that can be usefully used as a vaccine strain using the recombinant plasmid.

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

Still another object of the present invention is to provide a vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus as an active ingredient.

Another object of the present invention to provide a method for preventing foot and mouth disease using the vaccine composition.

In the present invention, the P1 gene region of the foot-and-mouth disease O type Manisa genome is substituted with the P1 gene of the foot-and-mouth disease SAT2 ZIM, and the 3B12 portion of the 3B region of the foot-and-mouth disease O type Manisa genome is replaced by the base sequence of 3B3 represented by SEQ ID NO: Provide the plasmid.

The present invention also provides a recombinant foot and mouth virus produced using the recombinant plasmid.

In addition, the present invention comprises the steps of (a) inserting a gene of foot-and-mouth disease O type Manisa into a recombinant vector; (b) replacing the 3B12 site of foot-and-mouth disease O type Manisa in the recombinant vector obtained in step (a); (c) in the recombinant vector obtained in step (b), replacing 3C 142 th amino acid cysteine in the P1 gene of the foot-and-mouth type O manisa with threonine (C142T); (d) In the recombinant vector obtained in the step (c), provides a method for producing a recombinant vector comprising the step of replacing the P1 gene of the foot-and-mouth disease O type Manisa with SAT2 ZIM gene.

The present invention also provides a vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus as an active ingredient.

The present invention also provides a method for preventing foot-and-mouth disease, comprising administering the recombinant foot-and-mouth virus to a subject other than a human.

Recombinant foot-and-mouth virus according to the present invention has the advantage that can be used more safely than the pathogenic virus to handle in the laboratory, such as disinfectant experiments or antiviral experiments by manipulating the gene part of the foot-and-mouth virus O virus.

In addition, by producing a vaccine using the recombinant foot-and-mouth virus according to the present invention as an active ingredient, there is an effect that can be protected to SAT type II II type region virus.

In addition, the recombinant foot-and-mouth virus according to the present invention can be differentially diagnosed with the outdoor strain and thus can be effectively used for vaccine production.

Figure 1 shows a genomic schematic of recombinant foot-and-mouth virus expressing the defense antigen of foot-and-mouth SAT type 2 foot-and-mouth virus II regional ZIM vaccine strain.
Figure 2 shows a schematic diagram of the recombinant plasmid (pO-SAT2 ZIM) of the present invention is inserted into the foot-and-mouth disease O vaccine strain Manisa nucleotide sequence encoding the protective antigen protein of SAT2 ZIM (region II).
FIG. 3 shows the newly prepared SAT2 ZIM virus particles by introducing the recombinant plasmid of FIG. 2 into the cells through an electron micrograph.
Figure 4 confirms that the pathogenicity of recombinant foot-and-mouth disease virus is reduced by measuring mortality after inoculation into mammalian mice with the SAT2 ZIM-wild type and the recombinant foot-and-mouth virus (SAT2 ZIM-R) of the present invention.
Figure 5 confirms that the antigenic protein is expressed in cells infected with the recombinant foot-and-mouth virus (SAT2 ZIM-R) of the present invention (Svanova kit, UK; SP band is band formation, C is experimental control, 375 ng [ 1/40 dose] can be detected).
FIG. 6 shows the survival rate and weight loss of mice through 7 days immunization (1.5 ~ 0.023 μg, bovine and pig 1/10 ~ 1/640 dose) challenge challenge (SAT2 ZIM wild type) The result is. According to these results, even when inoculated with a 0.093 μg (1/160 dose) vaccine, it was confirmed that complete protection against II type-type virus was possible.
Figure 7 shows the results of neutralizing antibody levels for antibodies immunized with serum obtained through the immunization test (vaccinates 0 and 4 weeks) in cattle (n = 5).
FIG. 8 shows the results of investigating neutralizing antibody levels of antibodies immunized with serum obtained through immunoassay (vaccination at 0 and 4 weeks) in pigs (n = 20).

The present invention relates to a recombinant plasmid having a nucleotide sequence of SEQ ID NO: 1 comprising a gene of foot-and-mouth disease O virus, wherein the P1 gene of the foot-and-mouth disease O type Manisa genome is substituted with a P1 gene of foot-and-mouth disease II region type SAT2-ZIM, It relates to a recombinant plasmid comprising the foot-and-mouth type O Manisa genome sequence, wherein the 3B12 site is substituted and the 142th amino acid cysteine in the 3C site is substituted with threonine.

In the present invention, "P1 gene" is a base sequence encoding the P1 protein, may include VP1, VP2, VP3 and VP4 sites, in the present invention, the site is a base sequence encoding the protein, the base without the protein coding Used as a concept involving sequences and genes.

The gene of foot-and-mouth disease O virus is characterized in that the entire gene of foot-and-mouth disease O vaccine strain Manisa. Part of the 3B1 region and 3B2 region may be substituted for the purpose of differential diagnosis with the outdoor strain among the entire gene region. Substitution sites of the 3B1 and 3B2 are 6501 to 6644 bp of the recombinant plasmid of the present invention represented by the nucleotide sequence of SEQ ID NO: 1 The site is preferably a 144 bp sequence represented by the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

In the present invention, "foot-and-mouth virus SAT2 type II regional type SAT2 ZIM strain" is a vaccine virus widely used in SAT2 type vaccine, and is known to be able to defend against virus of type II SAT2 type virus.

According to an embodiment of the present invention, the recombinant plasmid of the present invention may be substituted with threonine at the 142th amino acid cysteine of the 3C region of the foot-and-mouth type O Manisa genome (C142T).

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 SAT2 type ZIM strain is expressed.

According to another embodiment of the present invention, the present invention

(a) inserting a gene of foot-and-mouth type O Manisa into a recombinant vector; (b) replacing the 3B12 site of foot-and-mouth disease O type Manisa in the recombinant vector obtained in step (a); (c) in the recombinant vector obtained in step (b), substituting threonine for the 142th amino acid cysteine of the 3C region of the foot-and-mouth type O Manisa (C142T); And (d) replacing the P1 gene of foot-and-mouth disease O type Manisa with the P1 gene of foot-and-mouth disease SAT2-ZIM in the recombinant vector obtained in step (c).

According to another embodiment of the present invention, the present invention relates to a method for producing recombinant foot-and-mouth disease virus, comprising the step of introducing and propagating a recombinant vector prepared by the method.

As used herein, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are the most commonly used form of current vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising a screening label and (c) a restriction enzyme cleavage site into which foreign DNA fragments can be inserted. Although no appropriate restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods facilitates ligation of the vector and foreign DNA.

Recombinant vectors and recombinant foot-and-mouth virus of the present invention can be prepared by conventional genetic engineering, transformation method, it is possible to obtain an appropriate amount of virus in a continuous passage through the cell culture of the virus formed in a small amount.

The cells of the present invention are canine, feline, boar, bovine, deer, animal, giraffe, animal, pelican, camel, hippopotamus, horse, animal, maca, rhinoceros, weasel, rabbit, It may be derived from one or more cells selected from the group consisting of cells of rodents and primates, preferably goat tongue cells (ZZR) and hamster kidney cells (BHK-21), black goat kidney cells (BGK), porcine kidney cells (IBRS-2) and small kidney cells (LF-BK) can be used one or more selected from the group consisting of.

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

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

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

The vaccine composition of the present invention may be prepared by further including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, sucrose solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, 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 (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex, health of the individual. The range varies depending on the condition, diet, administration time, administration method, 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 virus to a subject other than human.

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

Example 1 Genetic Engineering for Differential Diagnosis of Foot-and-mouth Disease Outdoor Cells

The foot-and-mouth disease O type Manisa virus whole gene (Genbank AY593823.1) was amplified by PCR, and the recombinant plasmid pO-Manisa was prepared by inserting the O-Manisa gene into the plasmid (pBluescript SK II). After removing the 3B1 and 3B2 sites of the prepared pO-Manisa and then inserting by inserting the AB 3B3 and SAT 3B3 sequence synthesis (144 bps, SEQ ID NO: 2) pO for differential diagnosis with the outdoor strain -Mansia-3B plasmid was made.

Specifically, specific primers capable of amplifying the end gene of 3A and the start region of 3B3 based on cloning of the entire gene after cDNA preparation for O-Manisa virus using a random primer commonly used in cDNA synthesis (GenBank) PCR was carried out based on Accession No. AY593823.1, corresponding to 20 mers of each of the 5 'and 3' genes). PCR was performed at 98 ° C for 30 seconds at 98 ° C for 30 seconds at a dose of 5X buffer (FINNZYMES, 10µl), 10mM dNTPs (1µl), Phusion enzyme (2U / µl, 0.5µl), sterile distilled water (35.5µl). The reaction was carried out at 25 cycles of 30 ° C., 72 ° C. for 2 minutes 30 seconds, and final 72 ° C. for 10 minutes.

The ligation reaction (TAKARA Long Ligation kit) with the pO-Mansia synthesized by the PCR reaction was performed, and it was confirmed that it was properly cloned through the entire sequencing analysis. Table 1 below shows the amino acid sequences in which 3B1 and 3B2 of O-Manisa were removed and 3B3 of type A 3B3 and SAT were inserted.

NSP site manipulation Amino acid sequence Amino acid size Reference 3B12 deletion GPYAGPLERQKPLRVKTKLPQQEGPYAGPMDRQKPLKVRARAPVVKE (SEQ ID NO: 3) 47 aa - 3B33 addition GPYEGPVKKPVALKVKAKNLIVTEGPYEGPVKKPVALKVKAKAPIVTE (SEQ ID NO: 4) 48 aa A type and SAT type

Example 2. Pathogenicity Reduction Genetic Engineering

Attempts were first made to make infectious plasmids to produce foot-and-mouth virus with reduced pathogenicity. Carried out at a pO-Mansia-3B plasmid prepared in Example 1, the vector NEW ENGLAND BIOLABS ^ⓡ Inc. 142 the amino acid cysteine in the Gibson Assembly ^ⓡ Master Mix parts of pO-3C Manisa-3B gene using a threonine It was manipulated to substitute (C142T) (tg at the position 7140-7141 bp in the nucleotide sequence of SEQ ID NO: 1 to ac). After amplification using PCR, a self ligation method was used to secure the plasmid with the gene replacement.

Example 3 Construction of Recombinant Plasmid pO-SAT2 ZIM (FIGS. 1 and 2)

In the recombinant plasmid prepared in Example 2 (pO-Manisa 3B333 3Cmut), a gene encoding the P1 protein as a protective antigen was replaced with a gene encoding the P1 protein as a protective antigen of foot-and-mouth disease SAT1 virus BOT region type to insert a recombinant gene. Plasmid (pO-SAT2 ZIM) was prepared. The gene encoding the P1 protein was substituted as follows.

(1) First, RNA was extracted from wild type virus of SAT2 ZIM5 / 81 using Gibson Assembly® Cloning Kit (NEB) to synthesize cDNA, followed by plasmid (100ng / μl, 1μl), forward primer of SEQ ID NO: 5 (5 P1 gene of SAT2 ZIM5 / 81 virus was amplified by using '-AAGGTCCAGAAAAGGCTCAAGGGAGCAGGCCAGTCGTCACCA-3', 42mer) and reverse primer of SEQ ID NO: 6 (5'-GAGCAGGTCAAAATTTAGAAGCTGTTTGGCAGGTTTAACAAG-3 ', 42mer). At this time, the PCR is a 5X buffer (FINNZYMES, 10μl), 10mM dNTPs (1μl), Phusion enzyme (2U / μl, 1μl), sterile distilled water (35μl) after 30 seconds at 98 ℃, 10 seconds at 98 ℃, PCR was performed in 25 cycles of 72 ° C. 30 seconds, 72 ° C. 2 minutes 30 seconds, and then the reaction was performed at 72 ° C. for 10 minutes.

(2) PCR for removing P1 from the plasmid engineered with 3B and 3C sites (pO-Manisa 3B333 3Cmut) was carried out using V_Fwd (5'-ACTTCTAAATTTTGACCTGC-3), which is the forward primer of plasmid (100ng / μl, 1μl), SEQ ID NO: 7 20mer), V_Rev (5'-CTTGAGCCTTTTCTGGAC-3 ', 18mer), a reverse primer of SEQ ID NO: 8, was used. At this time, the PCR conditions are 5X buffer (FINNZYMES, 10μl), 10mM dNTPs (1μl), Phusion enzyme (2U / μl, 1μl), sterile distilled water (35μl) after 30 seconds at 98 ℃ 10 seconds, PCR was performed in 25 cycles of 65 ° C. 30 seconds, 72 ° C. 2 minutes 30 seconds, and then the reaction was performed at 72 ° C. for 10 minutes.

(3) P1 amplification products (200ng / μl, 1μl) and pO-Manisa 3B333 3Cmut of amplified product (200ng / μl, 1μl) removed from the SAT2 ZIM5 / 81, Gibson Assembly Master Mix (2X, 10μl) The reaction was carried out at 56 ° C. for 30 minutes in sterile distilled water (8 μl). Subsequently, pO-SAT2-ZIM plasmid (the entire sequence is shown in SEQ ID NO: 1) of the present invention was prepared by transformation into competent cells included in the Gibson Assembly® Cloning Kit. Finally, pO-Manisa 3B333 was subjected to sequencing. It was confirmed that the P1 site of the 3C mut plasmid was replaced with the P1 site of SAT2. The structure and schematic diagram of the produced recombinant plasmid pO-SAT2-ZIM are shown in FIGS. 1 and 2, respectively.

Example 4 Preparation of Recombinant Foot-and-mouth Virus Using the Plasmid of the Invention (FIG. 3)

Recovery of recombinant foot-and-mouth virus was performed by reacting the obtained recombinant plasmid (pO-SAT2 ZIM) with a restriction enzyme to prepare a gene into a single fragment, and transforming lipofectamine into BHKT7-9 cells (cell line expressing T7 RNA polymerase). Purified DNA was introduced using reagents (Life Technologies) and cultured for 2 to 3 days to ensure the recombinant virus formed. Thereafter, the secured virus was inoculated via serial passage in ZZ-R (goat fetal tongue) cells or BHK-21 (young hamster kidney) cells to propagate the virus. Thereafter, it was confirmed through the electron micrograph of FIG. 3 that the recombinant virus was remodeled in the cells infected with the foot and mouth virus.

Example 5 Immunogenicity in Animals of Constructed Vaccines (FIGS. 4-8)

0.5 ml of the foot-and-mouth virus virus (15 μg / dose) purified for animal experiments using the virus prepared as described above was mixed with 0.5 ml of the adjuvant containing the oil adjuvant ISA206 to prepare a double oil. The vaccine thus prepared was inoculated with 0.1 mL, 2-3 months old pigs, and 1 mL in cattle 6 months old or older in mice without foot-and-mouth antibody (C57BL / 6). (FIG. 6), pigs and cows were vaccinated at 0 and 4 weeks, and blood was collected for antibody measurement at each time, and neutralization test (based on the OIE manual standard test method) for the vaccine strain was performed to measure the SAT2 antibody. Was performed to determine antibody levels (FIGS. 7 and 8).

Although described with reference to the preferred embodiments and test examples of the present invention as described above, those skilled in the art without departing from the spirit and scope of the present invention described in the claims below It will be understood that various modifications and variations can be made in the present invention.

By providing a foot-and-mouth vaccine using the recombinant foot-and-mouth virus according to the present invention, which has a broad protective antigen expression ability of foot-and-mouth disease SAT2 serotype, it can contribute to the treatment and / or prevention of foot-and-mouth disease.

<110> Animal and plant quaratine agency <120> Recombinant foot-and-mouth disease virus expressing protective          antigen of type SAT2 ZIM <130> 17-10921 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 11114 <212> DNA <213> Artificial Sequence <220> <223> Full DNA sequence of pO-SAT2 ZIM <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 gggagcggga cagtcatcac cggctacagg gtcacagaat cagtctggta 2400 acactggaag cataataaac aactactaca tgcaacagta ccagaactca atggacaccc 2460 agcttggcga caacgccatt tcgggtgggt ccaacgaggg cagcacggac accacgtcca 2520 ctcacaccaa caacactcaa aacaacgatt ggttttccaa attggcccag tcggctatct 2580 caggactctt cggagccctg ttggctgaca agaaaactga ggagaccact ctgcttgagg 2640 accgcatcgt gacaacaaga cacggtacga ccacctccac cacgcaaagt tccgttggca 2700 tcacttacgg gtatgctgac gccgactcgt ttcggcccgg gcctaacacg tccgggttgg 2760 agacacgtgt caaacaggca gagcgattct tcaaagaaaa actttttgat tggacatcag 2820 acaaaccatt cggcacactg tacattttgg agttgcccaa ggaccacaaa gggatctatg 2880 ggagcctgac tgagtcttac gcttacatgc gtaatggctg ggacgtccag gtttctgcca 2940 cgagcacgca attcaacggc ggttcgcttc tcgtggccat ggtcccagag ctgtgctcat 3000 tgaagagcag agaagagttt cagcttactc tgtacccaca ccagttcatc aacccacgga 3060 ccaacaccac tgcacacatc caggttccct acctgggtgt gaacaggcac gaccaaggta 3120 agcgccacca ggcgtggtct ctggttgtca tggttctcac gcctcttacc accgaggcac 3180 agatgaactc cgggacagtt gaggtatacg ccaacatcgc cccgacgaac gtgttcgttg 3240 ctggcgaaaa gcccgcaaag caaggcatca ttccagttgc ttgctcagac ggctatggcg 3300 ggttccaaaa cactgacccg aagaccgcag acccgatcta cggttacgtc tacaacccgt 3360 ctcggaatga ctgtcacggt aggtactcca acctattgga cgtcgccgag gcgtgtccca 3420 cccttttgaa cttcgacggg aaaccgtacg tagtgacaaa gaacaatggt gataaagtta 3480 tgacctgttt tgacgtggca ttcactcaca aggtacacaa gaacaccttc ctggcgggtc 3540 ttgcggacta ctacacgcag taccaaggct cattgaacta ccacttcatg tacacaggtc 3600 ccacacacca caaagcaaag ttcatggtgg cgtacatccc accgggtgtg aacacagacg 3660 aactgccaaa gacccccgag gcggcggcgc actgctacca ctcggagtgg gacaccggac 3720 tgaactccca gttcacgttc gctgtgccct acgtctctgc cagtgacttc tcctacaccc 3780 acacagacac gcccgcaatg gcgaccacca acggatgggt ggcggtttac caagtgaccg 3840 acacccacac ggcggaggca gccgttgttg tgtctgtgag cgccggaccc gatctggagt 3900 tccgattccc gattgacccg gtgcggcaga ccacatcgtc aggtgaggga gcagacgtag 3960 tcacgactga tccctccacc cacggtgggt ctgtgacgga gaagaggcgc atgcacaccg 4020 atgtcgcctt cgtcatggac aggttcaccc acgtccacac caaccagact agcacagtga 4080 ttgacttgat ggacaccaat gagaagaccc ttgtgggtgc gttgctccgt gcttccacct 4140 actacttctg tgacatggaa attgcttgcg ttggcaaaca cgcacgcgtg tggtggcaac 4200 ccaacggtgc gccgcggaca acccagctcc gcgacaaccc aatggtgttc tcacacaaca 4260 acgtcacacg ctttgccctg ccgttcactg caccacaccg cctcctgtcc accaggtaca 4320 acggtgaatg caagtacaca cagacatcaa ccgccatccg tggcgaccgt gctgtgttgg 4380 cagccaaata cgcaaatgca aagcatgagc ttccctccac cttcaacttc gggtacttga 4440 ccgccgacga accagtcgac gtttattacc ggatgaagag gactgagctc tactgtccaa 4500 gagcccttct ccctgcttat gaccaccaaa gcagggacag gttcgacgcc cccattggcg 4560 tcgagaaaca acttctaaat tttgacctgc tcaaattggc gggagatgtg gagtccaacc 4620 ctgggccctt cttcttctcc gacgtcaggt caaatttctc aaaactggta gaaaccatca 4680 atcagatgca ggaggacatg tcaacaaaac acgggcctga ctttaaccgg ttggtgtccg 4740 catttgagga attggccact ggagtgaagg ctatcagggc cggtctcgac gaggccaaac 4800 cctggtacaa actcatcaag ctcctgagcc gcttgtcgtg catggccgct gtagcagcac 4860 ggtcaaagga cccagtcctt gtggccatca tgctggctga caccggtctt gagattctgg 4920 acagcacctt tgtcgtgaag aagatctccg actcgctctc cagtctcttt cacgtgccgg 4980 cccccgtctt cagtttcgga gccccgattc tgttggccgg gttggtcaaa gtcgcctcga 5040 gtttcttccg gtccacaccc gaagaccttg agagagcaga aaaacagctc aaagcacgtg 5100 acattaacga catattcgcc attctcaaga acggcgagtg gctggtcaag ctgatccttg 5160 ccatccgcga ctggatcaaa gcgtggatcg cctcagaaga aaagtttgtc accatgacgg 5220 acttggtgcc tggtatcctt gaaaagcagc gggatctcaa cgacccgagt aagtacaagg 5280 aagccaagga gtggctcgac aacgcgcgcc aggcgtgttt gaagagcggg aacgttcaca 5340 ttgccaattt gtgcaaagtg gtcgccccgg cacccagcaa gtcgagaccc gaacccgtgg 5400 tcgtttgcct ccgcggcaaa tccggccagg ggaagagttt ccttgcgaac gtgctcgcgc 5460 aagcaatctc cacccacttc accggcagaa ctgattcggt ttggtactgc ccgcctgacc 5520 ctgaccactt cgacggttac aaccagcaga ccgttgtcgt gatggacgat ttgggccaga 5580 accccgatgg caaggacttc aagtacttcg cccagatggt ttcgaccacg gggttcatcc 5640 cgcccatggc ctcgcttgag gacaaaggca agcctttcaa cagcaaagtc atcattgcta 5700 ccaccaacct gtactcgggt ttcaccccga gaacaatggt gtgtcctgac gcgctgaacc 5760 ggaggttcca ctttgacatc gacgtgagtg ccaaggacgg gtacaaagtt aacaacaaat 5820 tggacataat caaagctctt gaagacaccc acaccaaccc agtggcgatg ttccaatacg 5880 actgtgccct tctaaacggt atggcagttg aaatgaagag aatgcaacag gatatgttca 5940 agcctcaacc acccctccag aacgtgtacc aactcgttca cgaggtgatt gaacgggtcg 6000 agctccacga gaaggtgtcg agccacccga ttttcaaaca gatatcaatt ccttcccaaa 6060 agtctgtgtt gtacttcctc attgagaaag gccaacacga agcagcaatt gaattctttg 6120 agggaatggt gcatgactcc atcaaggaag agctccggcc cctcatccaa cagacctcat 6180 ttgtgaaacg cgcttttaag cgcctgaagg aaaactttga gactgttgcc ctgtgtttga 6240 ctcttttggc aaacatagtg atcatgatcc gcgagactcg caagagacaa cagatggtgg 6300 acgatgcagt gaatgactac attgagaagg caaacatcac cacagatgac aagactcttg 6360 acgaggcgga aaagaaccct ctagagacca gcggtgccag cactattggt ttcagagaga 6420 gaactctccc ggggcacaag gcgagcgatg acgtgagctc cgagcccgcc aaacccgtgg 6480 aggaccgacc acaagctgaa ggaccttacg agggaccggt gaagaagcct gtcgctttga 6540 aagtgaaagc taagaacttg attgtcactg aggggccata tgaaggacca gtgaagaaac 6600 ctgtcgcttt gaaagtgaaa gcaaaagccc cgattgtcac tgaaggaccc tacgagggac 6660 cggtgaagaa gcctgtcgct ttgaaagtga aagccaagaa cttgattgtc actgagagtg 6720 gtgccccacc gaccgacttg cagaagatgg tcatgggcaa cactaagcct gttgagctca 6780 tcctcgacgg gaagacggta gccatctgct gtgctaccgg agtgtttggc actgcctacc 6840 tcgtacctcg tcacctcttc gcggagaagt acgacaagat aatgttggac ggtagagcca 6900 tgacagacag tgactacaga gtgtttgagt ttgagattaa agtaaaagga caggacatgc 6960 tctcagacgc tgcactcatg gtgcttcacc gtgggaaccg cgtgagagac atcacgaaac 7020 attttcgtga cacagcaaga atgaagaaag gcacccccgt tgtcggtgtg atcaacaacg 7080 ccgacgttgg gagactgatt ttctctggag aggcccttac ctacaaagac attgtagtga 7140 ccatggatgg agacaccatg ccgggcctgt ttgcctacag agccgccacc aaggctggtt 7200 actgcggggg agccgttctc gccaaggacg gagccgacac attcatcgtt ggcacccact 7260 ccgcaggtgg taacggagtt ggatactgct cgtgcgtgtc caggtccatg ctcctgaaaa 7320 tgaaggcaca cattgaccct gaaccacacc acgaggggtt gattgttgat accagagatg 7380 tggaagagcg cgtgcatgtc atgcgtaaaa ccaagcttgc acccaccgtg gcacacggtg 7440 tgtttaaccc tgaatttggt cccgctgcct tgtccaacaa ggacccgcgg ctgaacgaag 7500 gggttgtcct cgatgaagtc atcttctcca aacacaaggg agacacgaaa atgtctgagg 7560 aggacaaagc gctgttccgc cgctgcgctg ccgactacgc gtcgcacttg cacagcgtgc 7620 tggggacggc aaatgcccca ttgagcatct atgaggccat caaaggcgtc gacgggctcg 7680 atgccatgga gccggacacc gcgcccggcc tcccctgggc cctccagggg aaacgccgtg 7740 gtgcgttgat tgacttcgag aacggcacgg tcggacccga agtcgaggct gccctaaagc 7800 tcatggagaa aagagagtac aaatttgctt gccagacctt cctgaaagac gagattcgtc 7860 cgatggaaaa agtacgtgct ggcaagactc gcattgtcga cgttttgccc gtggaacaca 7920 ttctttacac caggatgatg attggcagat tctgtgctca aatgcacaca aacaatggac 7980 cgcagattgg ctcagcggtc ggttgcaatc ctgatgttga ttggcaaaga tttggcacac 8040 attttgctca gtacagaaac gtgtgggatg tggactattc ggcctttgat gctaaccact 8100 gcagtgacgc aatgaacatc atgtttgagg aggtatttcg cacagacttc ggtttccacc 8160 caaatgctga gtggattctg aagactcttg tgaacacgga gcacgcctat gagaacaaac 8220 gtatcactgt tgagggcggg atgccgtctg gctgttccgc gacaagcatc atcaacacaa 8280 ttttgaacaa catttatgtg ctctacgctc ttcgtagaca ctatgaggga gttgagctgg 8340 acacctacac catgatctcc tacggagatg acatcgtggt tgcaagtgac tacgatctgg 8400 attttgaggc tctcaaaccc cacttcaaat ctcttggtca aaccatcact ccagctgaca 8460 aaagcgacaa aggttttgtt cttggtcact ccattaccga tgtcactttc ctcaaaagac 8520 acttccacat ggactatgga actgggtttt acaaacctgt gatggcctca aagaccctcg 8580 aggccattct ctcctttgca cgccgtggga ccatacagga gaagttgatc tccgtggcag 8640 gactcgccgt ccactcagga cctgacgagt accggcgtct ctttgagccc ttccagggtc 8700 tcttcgagat tccaagctac agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg 8760 acgcataatc cctcagatgt cacaattggc agaaagactc tgaggcgagc gacgccgtag 8820 gagtgaaaag cccgaaaggg cttttcccgc ttcctattcc aaaaaaaaaa aaaaaaaact 8880 agttctagag cggccgccac cgcggtggag ctccagcttt tgttcccttt agtgagggtt 8940 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct 9000 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 9060 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 9120 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 9180 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 9240 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 9300 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 9360 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 9420 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 9480 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 9540 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 9600 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 9660 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 9720 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 9780 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc 9840 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 9900 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 9960 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 10020 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 10080 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 10140 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 10200 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 10260 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 10320 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 10380 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 10440 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 10500 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 10560 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 10620 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 10680 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 10740 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 10800 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 10860 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 10920 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 10980 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 11040 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 11100 ccgaaaagtg ccac 11114 <210> 2 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of substitution region in 3B1 and 3B2 of O-Manisa <400> 2 ggaccttacg agggaccggt gaagaagcct gtcgctttga aagtgaaagc taagaacttg 60 attgtcactg aggggccata tgaaggacca gtgaagaaac ctgtcgcttt gaaagtgaaa 120 gcaaaagccc cgattgtcac tgaa 144 <210> 3 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of deletion region in 3B1 and 3B2 of O-Manisa <400> 3 Gly Pro Tyr Ala Gly Pro Leu Glu Arg Gln Lys Pro Leu Arg Val Lys   1 5 10 15 Thr Lys Leu Pro Gln Gln Glu Gly Pro Tyr Ala Gly Pro Met Asp Arg              20 25 30 Gln Lys Pro Leu Lys Val Arg Ala Arg Ala Pro Val Val Lys Glu          35 40 45 <210> 4 <211> 48 <212> PRT <213> Artificial Sequence <220> <223> Amino acid sequence of substitution region in 3B1 and 3B2 of          O-manisa <400> 4 Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys   1 5 10 15 Ala Lys Asn Leu Ile Val Thr Glu Gly Pro Tyr Glu Gly Pro Val Lys              20 25 30 Lys Pro Val Ala Leu Lys Val Lys Ala Lys Ala Pro Ile Val Thr Glu          35 40 45 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for P1 of SAT2 ZIM virus <400> 5 aaggtccaga aaaggctcaa gggagcaggc cagtcgtcac ca 42 <210> 6 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for P1 of SAT2 ZIM virus <400> 6 gagcaggtca aaatttagaa gctgtttggc aggtttaaca ag 42 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for P1-deleted-O-manisa virus <400> 7 acttctaaat tttgacctgc 20 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for P1-deleted-O-manisa virus <400> 8 cttgagcctt ttctggac 18

Claims (9)

The P1 gene region of the foot-and-mouth disease O type Manisa genome was substituted with the P1 gene of the foot-and-mouth disease SAT2 ZIM, and among the 3B sites of the foot-and-mouth disease O type Manisa genome, the 3B1 and 3B2 sites were substituted with the sequence of SEQ ID NO: 2, and the foot-and-mouth type O manisa In a recombinant plasmid in which the 142th amino acid cysteine of the genome 3C region gene is substituted with threonine (C142T),
The recombinant plasmid is a recombinant plasmid comprising the nucleotide sequence of SEQ ID NO: 1 (pO-SAT2 ZIM) to include foot-and-mouth disease O type Manisa genomic sequence. Recombinant foot-and-mouth virus produced using the recombinant plasmid of claim 1. The recombinant foot-and-mouth virus according to claim 2, wherein the recombinant virus is expressed in the defensive antigen of the SAT2 ZIM strain of foot-and-mouth disease SAT2 II region type. (a) inserting a gene of foot-and-mouth type O Manisa into a recombinant vector;
(b) replacing the 3B1 and 3B2 sites of foot-and-mouth disease O type Manisa with the sequence of SEQ ID NO: 2 in the recombinant vector obtained in step (a);
(c) in the recombinant vector obtained in step (b), substituting threonine for the 142th amino acid cysteine of the 3C gene of the foot-and-mouth type O manisa (C142T); And
(d) in the recombinant vector obtained in step (c), the method of producing a recombinant vector comprising the step of replacing the P1 gene region of the foot-and-mouth disease O type Manisa with the P1 gene of foot-and-mouth disease SAT2-ZIM. Method for producing a recombinant foot-and-mouth virus comprising the step of introducing and propagating the recombinant vector prepared in claim 4. The method of claim 5, wherein the cells are any one or more selected from goat fetal tongue cells and hamster kidney cells. A vaccine for foot-and-mouth disease prevention comprising the recombinant foot-and-mouth virus of claim 3 as an active ingredient. The vaccine composition for preventing foot-and-mouth disease according to claim 7, wherein the vaccine is a live vaccine, an attenuated vaccine, or a dead vaccine. A method for preventing foot-and-mouth disease, comprising administering the recombinant foot-and-mouth virus of claim 3 to a subject other than a human.

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