KR20230115478A - Recombinant foot-and-mouth disease virus and foot-and-mouth disease vaccine composition comprising the same - Google Patents

Abstract

The present invention relates to a recombinant foot-and-mouth disease virus and a foot-and-mouth disease vaccine composition comprising the same. The present invention can provide a recombinant foot-and-mouth disease virus which broadly protects against O serotypes prevalent in Asia or around the world, and a foot-and-mouth disease vaccine composition comprising the same.

Description

Recombinant foot-and-mouth disease virus and foot-and-mouth disease vaccine composition comprising the same}

The present invention relates to a recombinant foot-and-mouth disease virus and a foot-and-mouth disease vaccine composition comprising the same. It relates to a foot-and-mouth disease recombinant virus that broadly protects against serotype O and a foot-and-mouth disease vaccine composition comprising the same.

Foot and mouth disease (FMD) is a viral blistering disease that infects cloven-hoofed animals and is characterized by rapid replication and rapid spread. Due to its economic importance, this disease is classified as a very important animal disease by the World Organization for Animal Health (OIE), and when it occurs, it is impossible to trade between countries, so it acts as the most important factor in trade of livestock products.

The foot-and-mouth disease pathogen is a single-stranded bipolar RNA virus belonging to Piconaviridae and Aphthovirus genera, and is divided into 7 different serotypes (A, O, C, Asia1, SAT 1, SAT2, SAT3). being classified. In addition, since there are about 10 or more regional types of foot and mouth disease virus type O, if developed strictly, vaccines against these regional types should be developed and used as vaccine strains. However, in order to develop all vaccine strains, field strains must be continuously cultured in cells to have appropriate proliferative capacity, and this method has a disadvantage in that it takes a lot of time.

The foot-and-mouth disease virus O Manisa is already known as the most widely used vaccine strain, but it is known that it is difficult to defend against the virus that is currently prevalent in Asia due to its low vaccine matching rate. In the case of type O, it is important to use a vaccine strain capable of broad-spectrum protection against serotype O because viruses of various regional types are prevalent not only in Asia but also worldwide.

Registered Patent Publication No. 10-2009270

In order to solve the above problems, the present invention is intended to provide a foot-and-mouth disease vaccine composition comprising a recombinant foot-and-mouth disease virus capable of providing various regional defenses with a high matching rate in cattle and pigs and an antigen derived from the virus.

In addition, it is intended to provide a method for preventing and treating foot-and-mouth disease using the foot-and-mouth disease vaccine composition.

In order to achieve the above object, the present invention provides a recombinant virus based on foot-and-mouth disease virus O serotype and a foot-and-mouth disease vaccine composition comprising the same as an active ingredient.

In addition, a recombinant plasmid capable of producing the recombinant virus is provided.

In addition, the present invention provides a foot-and-mouth disease vaccine composition comprising the recombinant foot-and-mouth disease virus or an antigen isolated/purified from the recombinant foot-and-mouth disease virus and capable of protecting against various regional types.

In addition, a method for preventing and treating foot-and-mouth disease using the recombinant foot-and-mouth disease virus or a vaccine composition containing an antigen isolated and purified from the recombinant foot-and-mouth disease virus is provided.

In addition, a foot-and-mouth disease diagnostic kit comprising the foot-and-mouth disease recombinant virus or the foot-and-mouth disease virus antigen is provided.

In addition, a method for diagnosing foot-and-mouth disease using the foot-and-mouth disease diagnosis kit is provided.

The method for preventing or treating foot-and-mouth disease and the method for diagnosing foot-and-mouth disease are meant to be applied to ungulates, such as pigs, cows, goats, deer, and camels, excluding humans.

The recombinant foot-and-mouth disease viruses of the present invention (which may be referred to herein as O JC-VP1, O PA2-VP1) are based on the O serotype, and the VP1 of the O type Manisa (AY593823.1) is O/Jincheon/ It may be substituted with SKR/2014 (KU991728) or O PAK/44/2008 (GU384682).

Specifically, the recombinant foot-and-mouth disease virus according to the present invention may be a foot-and-mouth disease type O virus.

Specifically, O JC-VP1 and O PA2-VP1 are viruses purified from SEQ ID NO: 6 and SEQ ID NO: 7, respectively, and their base sequences are the 646th to 8864th sequence parts of SEQ ID NO: 6 and SEQ ID NO: 7, respectively. It may have a 646th to 8861st sequence.

In addition, the recombinant foot-and-mouth disease virus of the present invention can be administered together with an adjuvant that induces an increase in immunity.

As used herein, the term "plasmid" refers to a DNA preparation containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. Once transformed into a suitable host, the plasmid can replicate and function independently of the host genome or, in some cases, can integrate into the genome itself. As the plasmid is currently the most commonly used form of vector, "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) an origin of replication to ensure efficient replication, including several hundred plasmid vectors per host cell, and (b) a host cell transformed with the plasmid vector to be selected for selection. It has a structure including a selection marker and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cleavage site does not exist, the vector and the foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method.

The recombinant vector and recombinant foot-and-mouth disease virus of the present invention can be prepared by conventional genetic manipulation or transformation, and an appropriate amount of virus can be obtained by successive passage of the virus formed in small amounts through cell culture.

Cells used for cell culture are canines, felines, wild boars, bovines, deer, giraffes, peccaries, camels, hippopotamuses, horses, tapirs, rhinos, weasels , It may be derived from one or more cells selected from the group consisting of rabbits, rodents and primates, preferably goat tongue cells (ZZ-R) and hamster kidney cells (BHK-21), black goat kidney cells (BGK ), at least one selected from the group consisting of pig kidney cells (IBRS-2) and bovine kidney cells (LFBK).

The foot-and-mouth disease vaccine composition of the present invention contains, as an active ingredient, the foot-and-mouth disease recombinant virus of the present invention or an antigen isolated and purified from the recombinant virus.

A vaccine comprising the vaccine composition may be a live vaccine, an attenuated vaccine, or a killed vaccine.

The foot-and-mouth disease recombinant virus or a vaccine composition containing an antigen isolated and purified from the recombinant virus may be administered at a dose of 1/2560 to 1/10 dose, preferably at a dose of 1/640 to 1/10 dose. can be administered.

In addition, the vaccine composition may additionally include diluents or excipients such as carriers, fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are generally acceptable in the art.

In addition, the vaccine composition may be administered (or injected) to a subject in various forms. Administration may be performed by any one method selected from the group consisting of subcutaneous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, nasal administration, oral administration, transdermal administration and oral administration.

In addition, the present invention can provide a method for preparing the recombinant foot-and-mouth disease virus, and the method includes the following steps;

(a) inserting the gene of foot-and-mouth disease O serotype Manisa into a plasmid;

(b) substituting the VP1 site of foot-and-mouth disease O serotype Manisa in the recombinant plasmid obtained in step (a); and

(c) isolating and purifying the recombinant plasmid obtained in step (b).

The present invention relates to a recombinant foot-and-mouth disease virus and a foot-and-mouth disease vaccine composition comprising the same. A foot-and-mouth disease recombinant virus that broadly protects against serotype O and a foot-and-mouth disease vaccine composition comprising the virus can be provided.

Figure 1a shows a schematic diagram of foot-and-mouth disease recombinant viruses according to the present invention, including JC VP1 with O1 Manisa (VP4, 2, 3) and O/SKR/Jincheon/2014 (VP1).
Fig. 1b shows a schematic diagram of a foot-and-mouth disease recombinant virus according to the present invention comprising PA2 VP1 with O1 Manisa (VP4, 2, 3) and O PA-2, O PAK/44/2008 (VP1).
Figures 1c and 1d show the detection amount of FMDV inactivated antigen (PBM kit) using an O-type Rapid Test Kit, and Figures 1e and 1f show transmission electron microscopy results of FMDV vaccine strains JC-VP1 and PA2-VP1, respectively.
Figures 2a and 2b are respectively inoculated with the experimental vaccines (JC-VP1) and (PA2-VP1) at 1/10 (1.5 μg), 1/40, 1/160, and 1/640 doses for the three serotypes. It shows the result of defense ability.
Figure 3 shows the immunogenicity (neutralizing antibody and ELISA antibody) test results after vaccination with PA2-VP1 and JC-VP1 vaccines in cattle and pigs, (A) is JC-VP1 in cattle, (B) is Results for PA2-VP1 in cattle, (C) for JC-VP1 in pigs, and (D) for PA2-VP1 in pigs.
Figure 4 shows the results of PA2-VP1 and JC-VP1 vaccine matching tests in cattle and pigs, (A) is the matching result of JC-VP1 in cattle and pigs, (B) is PA2-VP1 in cattle and pigs This is the VP1 matching result.
Figures 5a to 5c are the results of evaluating the protective ability in pigs against the three regional types of the test vaccines O JC-VP1 and PA2-VP1, respectively. 5b shows the attack against three regional viruses after O-JC-VP1 vaccination, and FIG. 5c shows the results of attack against three regional viruses after O-PA2-VP1 vaccination.
Figure 6 shows the comparison values of neutralizing antibodies for each virus according to the type of vaccine candidate in swine. Neutralizing antibody comparison value, (C) is neutralizing antibody comparison value for O TWN (O taiwan 97) of vaccine candidates, (D) is O JC-VP1 vaccine change in the three attacking virus antibodies, (E) is O JC2 -O BE attack virus antibody change according to VP1 vaccine antigen amount change, (F) is 3 attack virus antibody change for O PA2-VP1 vaccine, (G) is O JC attack virus antibody change according to O PA2-VP1 vaccine antigen amount change is the result of change.
Figure 7 shows a schematic diagram of the plasmid sequence for the production of recombinant virus of O JC-VP1.
Figure 8 shows a schematic diagram of the plasmid sequence for the production of O PA2-VP1 recombinant virus.

Hereinafter, the present invention will be described in detail by examples and experimental examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following Examples and Experimental Examples.

<Example>

1. Recombinant Plasmid Preparation

Chimeric viruses were prepared as described in a previous study (Lee et al., 2017) (Prior document: Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses).

The remaining sequences except for the VP1 region in the plasmid (O1manisa) backbone sequence were amplified by PCR and synthesized based on O/PAK/44/2008 (Genbank No. GU384682) and O/SKR/JC/2014 (Genbank No.KX162590.1) Using one gene as a template, the VP1 region of PA2 and the VP1 region of JC were amplified using PCR, respectively, and then the genes were inserted, and oligonucleotide primers were used as follows.

VP1 of the viral structural protein was prepared by oligonucleotide primers based on O/SKR/JC/2014 (Genbank No.KX162590.1) and ME-SA (Panasia) O/PAK/44/2008 (Genbank No. GU384682) as follows. Used with:

* Primer sequence required for cloning

O1manisa_F (forward direction from 4552 of sequence information 1 for vector amplification):

CTTCTAAATTTTGACCTGCTCAAAT (SEQ ID NO: 8)

O1manisa_R (reverse direction from 3918 of sequence information 1 for vector amplification):

CTGTGTGCGAGCATCCACC (SEQ ID NO: 9)

PA2_VP1_F (5' beginning of SEQ ID NO: 6): ACCACCTCCACAGGTGAGTCAG (SEQ ID NO: 10)

PA2_VP1_R (3' part of SEQ ID NO: 6): CTGTTTCACAGGTGCCACTATC (SEQ ID NO: 11)

JC_VP1_F (5' start of SEQ ID NO: 3): ACCACTTCGACAGGCGAGTCG (SEQ ID NO: 12)

JC_VP1_R (3' part of SEQ ID NO: 3): CTGCTTTACAGGTGCCACTAT (SEQ ID NO: 13),

O JC-VP1 and O PA2-VP1 recombinant viruses were prepared as follows.

(1) Production of O JC-VP1 recombinant virus

To prepare O JC-VP1 recombinant virus, pOmanisa-3B333 vector sequence (recombinant plasmid vector backbone for O JC-VP1 construction (sequence engineered by replacing 3B ₁ B ₂ B ₃ with 3B ₃ B ₃ B ₃ in O Mansia) was inserted into the pBluescript II vector to prepare a recombinant plasmid of SEQ ID NO: 1. Then, the O1manisa_VP1 sequence (SEQ ID NO: 3) corresponding to the 3919th to 4551st sequences of SEQ ID NO: 1 was converted into the VP1 sequence of O Jincheon strain of SEQ ID NO: 4. By substitution, the recombinant plasmid of SEQ ID NO: 6 was completed.

(2) Production of O PA2-VP1 recombinant virus

To prepare the O JC-VP1 recombinant virus, pO Mansia: The recombinant plasmid of SEQ ID NO: 2 was prepared by inserting the O Mansia sequence into the pBluescript II vector. Then, the recombinant plasmid of SEQ ID NO: 7 was completed by replacing the O1manisa_VP1 sequence corresponding to the 3919th to 4551st sequences of SEQ ID NO: 2 with PA2_VP1 of SEQ ID NO: 5.

2. Cell culture and virus recovery

ZZ-R 127 cells were maintained with Dulbecco's Modified Eagle's Medium/F12 and BHK-21 cells were maintained with Dulbecco's Modified Eagle's Medium (Corning). 10% fetal bovine serum (Paisley, UK), 100 U/mL penicillin and 100 mg/mL streptomycin were added to the culture. BHK-T7-9 cells were cultured in 5% fetal bovine serum (Gibco BRL, Paisley, UK) and 10% Tryptose Phosphate Broth (Sigma-Aldrich, St. Louis, MO, USA). BHK-21 suspension cells were maintained in CD BHK-21 production medium (Gibco BRL, Paisley, UK) under 5% CO2 conditions.

The recovered virus was obtained using the same procedure as in the previous study (Lee et al., 2017). The recombinant plasmid was linearized by treatment with restriction enzyme Spel (NEB, Beverly, USA), and the infectious recombinant virus in BHK-T7-9 cells was transformed using Lipofectamine 3000 (Invitrogen, Carlsbad, USA), and this linearized plasmid was converted into a virus. was allowed to recover. The obtained recombinant virus was cultured and passaged at 37°C for 48 to 72 hours, and the virus was harvested with three freeze-thaw cycles and infected with the chimeric virus into fresh ZZ-R 127 cells. All experiments with FMDV or virus-associated animals were performed at Biosafety Level 3 (BSL-3).

3. Virus Purification for Vaccine Antigens

The FMDV antigen was described in a previous study (Ko et al., 2019) (prior document title: Improved foot-and-mouth disease vaccine with O PanAisa-2 strain protect pigs against O/Jincheon/SKR/2014 originated from South Korea) It has been refined through some modifications. Briefly, virus grown in BHK21 suspension cells was harvested by freezing and thawing when complete cytopathic effect was observed. After inactivating the virus with binary ethyleneimine (0.003N) for 24 hours at 26℃, cell debris was removed. Inactivated viral antigens were precipitated with 7.5% PEG 6000 and 2.3% NaCl at 4° C. overnight, and the pellet was resuspended in Tris-KCl to a final concentration of 200 fold. Centrifugation was performed with a sucrose density gradient from 15 to 45. After centrifugation at 30,000 rpm for 4 hours in a SW41 rotor at 4, the amount of antigen was determined by spectrophotometric analysis at 259 nm. For electron microscopic analysis, the purified antigen was adsorbed onto a carbon-coated copper grid and visualized by transmission electron microscopy (Hitachi H7100FA, Tokyo, Japan). O JC-VP1 and O PA2-VP1 recombinant viruses are viruses purified from SEQ ID NO: 6 and SEQ ID NO: 7, respectively. confirmed to be

4. Vaccination and FMD Virus Challenge in Adult C57BL/6 Mice

An easy and quick method was used in mice to predict defense against various regional types of viruses prior to challenge testing in target animals. Seven-week-old C57BL/6 female mice were used. An experimental group (5 mice/group) was used. Antigens (1/10, 1/40, 1/160 and 1/640 dose groups (15 μg/dose) of vaccination in pigs) were mixed with ISA 206 (double oil emulsion). W/O/W type) and 10% aluminum hydroxide gel adjuvant were also mixed. Mice were challenged 7 days after intramuscular vaccination. O/VIT/2013 (ME-SA regional) virus after IP injection with 3 x 10^4 TCID50/0.1ml and O/Jincheon/SKR/2014 (SEA regional) and O/Taiwan/97 ( Cathay regional type) virus 1 x 10^5 TCID ₅₀ /0.1 ml challenged and observed for 7 days.

5. Immunogenicity testing of experimental vaccines for cattle and pigs

5-month-old cattle and 8-10-week-old pigs (n = 5) were inoculated with 15 μg/dose antigen mixed with ISA 206, saponin and aluminum hydroxide gel adjuvant. Bovine serum was collected on days 14, 28, 42, and 56 post-vaccination and porcine serum was collected on days 7, 14, 21, 28, 42, 56, and 84 post-vaccination. Pigs were boosted on day 28. Antibodies against structural proteins of FMDV in serum were detected using PrioCheck FMDV O (Prionics AG, Schlieren-Zurich, Switzerland). > 50 % Inhibition (%) values were reflected. Animals were considered to demonstrate an immune response as follows.

6. Virus neutralization test for bovine and porcine serum

Serum samples were inactivated by heat treatment at 56°C for 30 minutes. Cell concentration was adjusted to form a 70% monolayer and 2-fold serial dilutions of serum samples were prepared. Diluted serum samples were incubated with a viral titer of FMDV 100 TCID ₅₀ of the virus for 1 hour at 37°C. After 1 hour, the LFBK cell suspension was added to all 96 well plates. The cytopathic effect was checked after 2-3 days to determine the antibody titer, which was calculated as the Log10 of the reverse antibody dilution required to neutralize 100 TCID ₅₀ of the virus.

7. Challenge inoculation of pigs immunized with recombinant vaccine

Three different viruses (10^5.0 TCID ₅₀ /0.1 ml each) were injected intradermally into the footpad for challenge on day 28 post vaccination. Serum and oral swabs were collected 8 days after viral challenge. Blood samples were also collected. Oral swabs were collected using the BD™ Universal Virus Delivery Kit (BD, USA) and clinical scores were determined by adding the distributed scores as follows. Clinical observations were made daily after challenge. Clinical scores were calculated based on a maximum of 15 points using the following criteria: (a) hoof and foot follicles (1-2 points per foot); (b) Muzzle, labial, and lingual vesicles (one point for each affected area).

8. Detection of viremia in immunized pigs

Real-time (RT) PCR was performed with glandular serum and swab sampling: mouth and nose swabs were taken from experimental animals using swabs as follows. Total cellular RNA was extracted using the QIAcube HT (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. RT-PCR was performed using a one-step prime-script RT-PCR kit (Bioneer, Republic of Korea) according to the manufacturer's instructions. For virus quantification, CFX96 TouchTM Real-Time PCR Detection System (Bio-Rad, USA) was used.

<Experimental Example 1> Confirmation of virus according to the present invention

Replaced the VP1 protein coding sequence that induces the most important antigenic mutation in the foot-and-mouth disease virus infectious plasmid into which the entire gene of foot-and-mouth disease virus O1 Manisa has been inserted with Jincheonju (O/SKR/Jincheon/2014), which was a problem in Korea in 2014 to secure the O-JC VP1 virus (FIG. 1a). In addition, O-PA2 VP1 virus was obtained by replacing VP1 with O PanAsia-2 (O/PAK/44/2008) (Fig. 1b).

The purified JC-VP1 and PA2-VP1 inactivated antigens were measured using a foot-and-mouth disease antigen simple kit. Control (C) showed the same line, and the line at the SP position was observed in proportion to the amount of antigen (Fig. 1c, 1d)

The antigen morphology of JC-VP1 and PA2-VP1 was observed using a transmission electron microscope, respectively (FIGS. 1e and 1f). It is confirmed that a new vaccine strain virus is created using genetic recombination technology, and it can be seen that it can be developed as an inactivated vaccine.

<Experimental Example 2> Evaluation of the protective ability of the vaccine according to the present invention

In order to test the protective ability of the new recombinant vaccine against three regional viruses, an experiment was conducted to measure PD50 using C57BL/6 female mice. Each target virus ME-SA (O Vet) 7 days after immunization of mice with JC-VP1, PA2-VP1 vaccine (1/10, 1/40, 1/160, 1/640 of the amount of antigen for the target animal) , SEA (O JC), and Cathay (O TWN97) viruses were challenged with 100LD ₅₀ titer, and the survival rate and weight loss were confirmed to confirm the effect of the challenge, that is, the protective effect.

Experimental results, in case of JC VP1 SEA (O/Jincheon/SKR/2014) challenge inoculation 55.7 mPD ₅₀ , ME-SA (O/VIT/2013) challenge inoculation 18 mPD ₅₀ , Cathay (O/Taiwan/97) At the time of challenge inoculation, 97 mPD ₅₀ was shown (FIG. 2a).

In addition, as a result of vaccination with PA2-VP1, SEA (O/Jincheon/SKR/2014) challenge vaccination 32 mPD ₅₀ , ME-SA (O/VIT/2013) virus challenge vaccination 128 mPD ₅₀ or more, Cathay (O/Taiwan/97) challenge showed 90.5 mPD ₅₀ (Fig. 2b).

<Experimental Example 3> Immunogenicity evaluation of the vaccine according to the present invention

In the case of cattle immunity test, it is known as the most standard animal test method among animals, and in general, 3-4 weeks after immunization with vaccine, it was confirmed through 2D neutralization test whether the vaccine matches the field strain with blood. Therefore, in the case of immunity experiments in cattle, it was prepared in the same way as the vaccines tested in mice and pigs, and antibody levels were compared by inoculating 1ml/dose. After immunizing cows with PA2-VP1 and O JC-VP1 only once, the antibody level was confirmed up to 8 weeks. As a result, the antibody level at 14 days after inoculation showed a very high level for all three antigens and was maintained at a high titer during the test period. In the case of PA2-VP1, neutralizing antibody reactivity was excellent in 1 week in cattle. It was confirmed that high antibody levels were maintained with only the first inoculation. In the case of JC-VP1, high neutralizing antibodies were maintained in the cows after 2 weeks, and high antibody levels were maintained even with the first inoculation (Fig. 3 a, b).

Evaluating the immunity of vaccines through swine immunogenicity testing is an important criterion for evaluating the effectiveness of vaccines in the field, especially in Korea. Therefore, in order to determine the efficacy of the vaccine through the experiment in pigs, we tried to evaluate the effect through an immunological experiment with 5 animals per group, and after inoculating the vaccine (1ml/dose) used for immunity experiments in mice and cows once After 4 weeks, the inoculation was performed twice, blood was collected every week until 4 weeks, and then blood was collected at intervals of 2 weeks to confirm the immunoreactivity (Fig. 3c, d).

<Experimental Example 4> Matching evaluation in cattle and pigs of the vaccine according to the present invention

This is the result of a vaccine matching experiment (r1 = field neutralization test titer / vaccine strain neutralization test titer) through a 2D neutralization test with blood 28 days after immunization.

As a result, blood on day 28 showed excellent immunogenicity in both groups vaccinated with the two vaccines, but matching results (r1) showed different characteristics. In the case of PA2-VP1, SEA was partially matched in pigs, but matched to ME-SA and Cathay. Reactivity was low in most SEA geotypes. The r1 index was higher in pigs than in cattle (Fig. 4a).

In the case of JC-VP1, most matches were made in pigs, but SEA was partially low in cattle and ME-SA was not matched. The reactivity of ME-SA is low. The r1 index is higher in pigs than in cattle (Fig. 4b). In the foot-and-mouth disease vaccine manual of the OIE (World Organization for Animal Health), it is a standard method to perform a matching test based on bovine immune serum so far, so it is a result that cannot be ignored.

<Experimental Example 5> Evaluation of the protective ability of the vaccine according to the present invention in pigs against the O type 3 regional types

As a result of challenge inoculation with SEA, ME-SA, and Cathay type among the negative control group, and measurement of viremia, symptoms tended to appear most rapidly when inoculated with Cathay type (FIG. 5a). When challenged with BE (ME-SA) type in the group vaccinated with JC-VP1, 50% protection was shown (ME-SA #4~7), but the amount of antigen was increased by 20ug (ME-SA #12~15) Therefore, vaccination could raise the protection rate to 100% (FIG. 5b). In the case of the PA2-VP1 vaccine, 100% protection was confirmed in both subjects vaccinated with 1 dose (15ug) and SEA #12-15 subjects vaccinated with 1.3 dose (20ug) (FIG. 5c).

<Experimental Example 6> Evaluation of neutralizing antibodies in pigs of the vaccine according to the present invention

As expected, the heterologous VN titer was highest in the JC-VP1 vaccination group in the JC (SEA type) challenge group (Fig. 6a), and the same in the BE (ME-SA) challenge group. It was the highest in the group vaccinated with ME-SA type PA2-VP1 vaccine (Fig. 6b). When inoculated with TWN (Cathay), PA2-VP1 and JC-VP1 showed similar results (Fig. 6 c)

Compared to the increase in homologous VN titer from 14 dpv in the case of JC-VP1, it can be seen that the homologous VN titer rapidly increased in 7 dpv in the PA2-VP1 vaccination group. In the case of JC-VP1, after showing the highest value at 21 dpv, a decreasing trend was confirmed at 28 dpv. (d, f in Fig. 6)

Homologous VN titers tended to be proportional to the antigenic amounts of JC-VP1 and PA2-VP1. In particular, there was a significant difference at 2 dpc immediately after challenge in the JC-VP1 15ug and 20ug vaccination groups (Fig. 6 e, g). This is judged to be the basis for showing a 50% defense rate at 15ug, but a 100% defense rate at 20ug.

<110> Animal and Plant Quarantine Agency <120> Recombinant foot-and-mouth disease virus and foot-and-mouth disease vaccine composition comprising the same <130> 21-12107 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 11056 <212> DNA <213> Artificial Sequence <220> <223> Recombianant plasmid <400> 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aa atcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt ttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcg ctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 ggg atggct 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 tgtgcctc gg ctgtcgcccg aagcccgcct ttcacccccc 1020 ccccccccct aggttttacc gtcgttcccg acgttaatgg ggaaacaacc acaagcttaa 1080 caccgtcttg cccgacgtaa aagggctgca accaaaaagc ttgtgccgcc tttcccggcg 1140 ttaatgggag gtaaccacaa gacaaacctt cacccggaag taaaacggca acttcacaca 1200 gttttgcccg ttttcgtgag aaatgggccg tca acgcacg aaacgcgccg tcgcttgagg 1260 aggacttgta caaacacgat ctatgcaggt ttccacaact gacacaaacc gtgcaacttg 1320 aaaccccgcc tggtctttcc aggtctagag gggcgacatt ttgtactgtg cttgactcca 1380 cgctcggtcc actagcgagt gttagtagta gcactgttgc ttcgtagcgg agcatgatgg 1440 ccgtgggagc ttccccttgg taacaaggac ccacggggcc aaaagccacg tcctaccgga 1500 cccatcatgt gtgcaaaccc agcacggcaa ctttactgcg aaaaccactt taaggtgaca 1560 ctgatactgg tactcaatca ctggtgacag gctaaggatg cccttcaggt accccgaggt 1620 aacacgcgac actcgggatc tgagaagggg actggggctt ctttaaaagt gcccagt c ggtgagaag 1860 aaaaccttct actccagacc caacaaccac gacaactgct ggcttaacac cattctccag 1920 ttgttcaggt atgttgatga gcctttcttt gactgggtct acgactcgcc tgaaaacctc 1980 actcttgagg caatcaaaca gttggaagag acaaccggtc ttgagctgca cgagggtgga 2040 ccacccgctc tcgtcatctg gaacatcaaa cacttgcttc acaccggaat cggcactgcc c ggacccgtc cgacgttctg 2280 gtgtttgtcc cgtacgatca agaaccgctt aacggagagt ggaaaacaaa ggtccagaaa 2340 aggctcaagg gagccgggca atccagcccg gcaaccgggt cacagaacca atcaggcaac 2400 actgggagca tcatcaacaa ttactacatg cagcagtacc aaaactccat ggacacacaa 2460 cttggtgaca acgctacaag cggaggctca aacgaggggt ccacggacac aacctccacc 2520 cacacaacca acactcagaa caacg actgg ttctcgaagc tggccagttc cgctttcagc 2580 ggtcttttcg gcgctcttct cgccgacaag aaaaccgagg agaccactct tctcgaggac 2640 cgcatcctca ctactcgtaa cggacacacc acctcgacaa cccagtcgag cgttggagtc 2700 acgtac gggt atgcaacagc tgaggatttc gtgagcgggc caaacacctc tggtctcgag 2760 accagggttg cccaggcaga gcggttcttt aaaacccacc tgttcgactg ggtcaccagt 2820 gacccattcg gacggtgcca cctgctggaa cttccaactg accacaaagg tgtctacggc 2880 agcctgaccg actcgtatgc ttatatgagg aacggctggg atgttgaagt cactgcagtg 2940 ggaaaccagt tca atggagg atgcctgttg gtggccatgg tgccagaact ttgctccata 3000 cagaagaggg agctgtacca gctcacgctc tttcctcacc agttcatcaa ccctcggacg 3060 aacatgacag cacacatcac tgtgcccttt gttggcgtca accgttatga ccagtacaag 3120 gtac acaaac cttggaccct cgtggttatg gttgtagccc ccctgactgt caacagtgaa 3180 ggtgccccgc aaatcaaggt gtatgccaac atcgcaccta ccaacgtaca cgtcgcgggt 3240 gagttccctt ccaaagaggg gatcttccct gtggcttgca gcgatggtta tggcggtctg 3300 gtgaccactg acccgaaaac ggctgacccc gcttacggga aagtgtttaa ccccccccgc 3360 aacatgttgc cggggcggtt caccaatt tt cttgacgtgg ctgaggcgtg ccccacgttt 3420 ctccacttcg agggtgacgt gccatacgtg accacgaaga cggattcaga cagggtgctc 3480 gctcagttcg acttgtcttt ggcagcaaag cacatgtcga acaccttcct tgcaggtctc 3540 gcccagtact acacacag ta cagcggcacc atcaacctgc acttcatgtt cacagggcct 3600 actgacgcga aggcgcgtta catgattgcg tatgctcctc ctggcatgga accacctaaa 3660 acgccagagg cggctgccca ctgcattcat gctgaatggg acacagggtt gaactcaaaa 3720 ttcacatttt caatccctta cctttcggcg gctgattacg cttacacagc gtctgacact 3780 gctgagacca caaatgtaca gggatgggtt tgcctgttt c aaataacaca cgggaaagct 3840 gacggcgacg cactggtcgt tttggctagc gccggaaagg actttgagct gcgcctgccg 3900 gttgatgctc gcacacagac tacctccgcg ggcgagtcag ctgaccccgt gaccgccacc 3960 gttgagaatt acggtggcga gac acaggtc cagaggcgcc aacacacgga cgtctcattt 4020 atattagaca gatttgtgac agtgacacca aaagaccaaa ttaatgtatt ggacctgatg 4080 caaacccctg ctcacacttt ggtgggagca ctccttcgta ctgccactta ctatttcgct 4140 gacttagagg tggcagtgaa gcacaaggga aacctcacct gggtcccgaa cggggcgcct 4200 gaagcggcgt tggacaacac caccaaccca acagcttacc acaa ggcacc actcacccga 4260 cttgcactgc cttacacggc gccacaccgc gtgttggcta ctgtttacaa cgggaactgc 4320 aagtatggtg acggcacggt ggccaatgtg agaggtgacc tgcaagtgtt ggcccagaag 4380 gcggcgagag cgctgcctac ctccttcaac tacggtgcca ttaaagctac tcgggtgact 4440 gaactgcttt accgcatgaa gagggctgag acatactgcc cccggcctct tttggccatt 4500 cacccggacc aggctagaca caagcagaag attgtggcac cggtggaaca gcttctaaat 4560 tttgacctgc tcaaattggc gggagatgg gagtccaacc ctgggccctt cttcttctcc 4620 gacgtcaggt caaatttctc aaaactggta gaaaccatca atcagatgca gg aggacatg 4680 tcaacaaaac acgggcctga ctttaaccgg ttggtgtccg catttgagga attggccact 4740 ggagtgaagg ctatcagggc cggtctcgac gaggccaaac cctggtacaa actcatcaag 4800 ctcctgagcc gcttgtcgtg catggccgct gtagcagcac ggtca aagga cccagtcctt 4860 gtggccatca tgctggctga caccggtctt gagattctgg acagcacctt tgtcgtgaag 4920 aagatctccg actcgctctc cagtctcttt cacgtgccgg cccccgtctt cagtttcgga 4980 gccccgattc tgttggccgg gttggtcaaa gtcgcctcga gtttcttccg gtccacaccc 5040 gaagaccttg agagagcaga aaaacagctc aaagcacgg acattaacga catattcg cc 5100 attctcaaga acggcgagtg gctggtcaag ctgatccttg ccatccgcga ctggatcaaa 5160 gcgtggatcg cctcagaaga aaagtttgtc accatgacgg acttggtgcc tggtatcctt 5220 gaaaagcagc gggatctcaa cgacccgagt aagtacacagg aagcca agga gtggctcgac 5280 aacgcgcgcc aggcgtgttt gaagagcggg aacgttcaca ttgccaattt gtgcaaagtg 5340 gtcgccccgg cacccagcaa gtcgagaccc gaacccgtgg tcgtttgcct ccgcggcaaa 5400 tccggccagg ggaagagttt ccttgcgaac gtgctcgcgc aagcaatctc cacccacttc 5460 accggcagaa ctgattcggt ttggtactgc ccgcctgacc ctgaccactt cgacggtac 5 520 aaccagcaga ccgttgtcgt gatggacgat ttgggccaga accccgatgg caaggacttc 5580 aagtacttcg cccagatggt ttcgaccacg gggttcatcc cgcccatggc ctcgcttgag 5640 gacaaaggca agcctttcaa cagcaaagtc atcattgcta ccacca acct gtactcgggt 5700 ttcaccccga gaacaatggt gtgtcctgac gcgctgaacc ggaggttcca ctttgacatc 5760 gacgtgagtg ccaaggacgg gtacaaagtt cga ggtgatt gaacgggtcg agctccacga gaaggtgtcg 6000 agccacccga ttttcaaaca gatatcaatt ccttcccaaa agtctgtgtt gtacttcctc 6060 attgagaaag gccaacacga agcagcaatt gaattctttg agggaatggt gcatgactcc 6120 atcaaggaag agctcc ggcc cctcatccaa cagacctcat ttgtgaaacg cgcttttaag 6180 cgcctgaagg aaaactttga gactgttgcc ctgtgtttga ctcttttggc aaacatagtg 6240 atcatgatcc gcgagactcg caagagacaa cagatggtgg acgatgcagt gaatgactac 6300 attgagaagg caaacatcac cacagatgac aagactcttg acgaggcgga aaagaaccct 6360 ctagagacca gcggtgccag cactattggt tt cagagaga gaactctccc ggggcacaag 6420 gcgagcgatg acgtgagctc cgagcccgcc aaacccgtgg aggaccgacc acaagctgaa 6480 ggacccttacg agggaccggt gaagaagcct gtcgctttga aagtgaaagc taagaacttg 6540 attgtcactg aggggccata tgaa ggacca gtgaagaaac ctgtcgcttt gaaagtgaaa 6600 gcaaaagccc cgattgtcac tgaaggaccc tacgagggac cggtgaagaa gcctgtcgct 6660 ttgaaagtga aagccaagaa cttgattgtc actgagagtg gtgccccacc gaccgacttg 6720 cagaagatgg tcatgggcaa cactaagcct gttgagctca tcctcgacgg gaagacggta 6780 gccatctgct gtgctaccgg agtgtttggc act gtgg gaaccg cgtgagagac atcacgaaac attttcgtga cacagcaaga 7020 atgaagaaag gcacccccgt tgtcggtgtg atcaacaacg ccgacgttgg gagactgatt 7080 ttctctggag aggcccttac ctacaaagac attgtagtga ccatggatgg agacaccatg 7140 ccgggcctgt ttgcctacag agccgccacc aaggctggtt actgcggggg agccgttctc 7200 gccaaggacg gagccgacac attcatcgtt ggcacccact ccgca ggtgg taacggagtt 7260 ggatactgct cgtgcgtgtc caggtccatg ctcctgaaaa tgaaggcaca cattgaccct 7320 gaaccacacc acgaggggtt gattgttgat accagagatg tggaagagcg cgtgcatgtc 7380 atgcgtaaaa ccaagcttgc acccaccgtg gc acacggtg tgtttaaccc tgaatttggt 7440 cccgctgcct tgtccaacaa ggacccgcgg ctgaacgaag gggttgtcct cgatgaagtc 7500 atcttctcca aacacaaggg agacacgaaa atgtctgagg aggacaaagc gctgttccgc 7560 cgctgcgctg ccgactacgc gtcgcacttg cacagcgtgc tggggacggc aaatgcccca 7620 ttgagcatct atgaggccat caaaggcgtc gacgggctcg atgccatg ga gccggacacc 7680 gcgcccggcc tcccctgggc cctccagggg aaacgccgtg gtgcgttgat tgacttcgag 7740 aacggcacgg tcggacccga agtcgaggct gccctaaagc tcatggagaa aagagagtac 7800 aaatttgctt gccagacctt cctgaaagac gag attcgtc cgatggaaaa agtacgtgct 7860 ggcaagactc gcattgtcga cgttttgccc gtggaacaca ttctttacac caggatgatg 7920 attggcagat aatga acatc 8100 atgtttgagg aggtatttcg cacagacttc ggtttccacc caaatgctga gtggattctg 8160 aagactcttg tgaacacgga gcacgcctat gagaacaaac gtatcactgt tgagggcggg 8220 atgccgtctg gctgttccgc gacaagcatc atcaacaca tt ttgaacaa catttatgtg 8280 ctctacgctc ttcgtagaca ctatgaggga gttgagctgg acacctacac catgatctcc 8340 tacggagatg acatcgtggt tgcaagtgac tacgatctgg attttgaggc tctcaaaccc 8400 cacttcaaat ctcttggtca aaccatcact ccagctgaca aaagcgacaa aggttttgtt 8460 cttggtcact ccattaccga tgtcactttc ctcaaaagac acttccacat ggactatgga 8520 actgg gtttt acaaacctgt gatggcctca aagaccctcg aggccattct ctcctttgca 8580 cgccgtggga ccatacagga gaagttgatc tccgtggcag gactcgccgt ccactcagga 8640 cctgacgagt accggcgtct ctttgagccc ttccagggtc tcttcgagat tccaag ctac 8700 agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg acgcataatc cctcagatgt 8760 cacaattggc agaaagactc tgaggcgagc gacgccgtag gagtgaaaag cccgaaaggg 8820 cttttcccgc ttcctattcc aaaaaaaaaa aaaaaaacta gttctagagc ggccgccacc 8880 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc 8 940 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 9000 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 9060 tgcgttgcgc tcactgcccg ctttccagtc gggaaacct g tcgtgccagc tgcattaatg 9120 aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 9180 cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 9240 ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 9300 ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 9360 cccccctgac gagcatcaca aaaat cgacg ctcaagtcag aggtggcgaa acccgacagg 9420 actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 9480 cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 9540 tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 9600 gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 9660 caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 9720 agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 9780 tagaaggaca gtatttggta tctgcgctct gctgaagcca gtta ccttcg gaaaaagagt 9840 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 9900 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 9960 gtctgacgct cagtg gaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 10020 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 10080 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 10140 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 10200 acgggagggc ttaccatctg gccccagtgc tgcaatgata caataaac ca gccagccgga 10260 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 10320 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 10380 gctacaggca tcgtggtgtc acgctcgtcg t ttggtatgg cttcattcag ctccggttcc 10440 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 10500 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 10560 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 10620 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc t tgcccggcg 10680 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 10740 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 10800 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 10860 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 10920 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 10980 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 11040 ccccgaaaag tgccac 11056 <210> 2 <211> 11090 <2 12> DNA <213> Artificial Sequence <220> <223> Recombianant plasmid <400> 2 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 ttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctgg c aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg tag ggttgaa aggggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagttggact cacggcaccg 780 cgtggccatt ttagctgggt tgtgcggacg aac actgctt gcgcatctcg cgtgaccggt 840 tagtactctt accactatcc gcctacttgg tcgttagcgc tgtcctgggc actcttgttg 900 ggggctgttc aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 ccccccccct aggttttacc gtcgttcccg acgttaatgg ggaaaca acc acaagcttaa 1080 caccgtcttg cccgacgtaa aagggctgca accaaaaagc ttgtgccgcc tttcccggcg 1140 ttaatgggag gtaaccacaa gacaaacctt cacccggaag taaaacggca acttcacaca 1200 gttttgcccg ttttcgtgag aaat gggccg tcaacgcacg aaacgcgccg tcgcttgagg 1260 aggacttgta caaacacgat ctatgcaggt ttccacaact gacacaaacc gtgcaacttg 1320 aaaccccgcc tggtctttcc aggtctagag gggcgacatt ttgtactgtg cttgactcca 1380 cgctcggtcc actagcgagt gttagtagta gcactgttgc ttcgtagcgg agcatgatgg 1440 ccgtgggagc ttccccttgg taacaaggac ccacggggcc aaaagccacg tcctaccgga 1500 cccatcatgt gtgcaaaccc agcacggcaa ctttactgcg aaaaccactt taaggtgaca 1560 ctgatactgg tactcaatca ctggtgacag gctaaggatg cccttcaggt accccgaggt 1620 aacacgcgac actcgggatc tgagaagggggg actt ctttaaaagt gcccagttta 1680 aaaagcttct atgcctgaat aggcgaccgg aggccggcgc cttttcactg ttttactact 1740 gttttcatga atacaactga ctgtttcacc gccctgttac acgctctcag agagatcaaa 1800 acactgtttc ttttacggac acaaggaaag atggaattca cactttacaa cggtgagaag 1860 aaaaccttct actccagacc caacaaccac gacaactgct ggcttaacac cattctccag 1920 ttgttcaggt atgttgat ga gcctttcttt gactgggtct acgactcgcc tgaaaacctc 1980 actcttgagg caatcaaaca gttggaagag acaaccggtc ttgagctgca cgagggtgga 2040 ccacccgctc tcgtcatctg gaacatcaaa cacttgcttc acaccggaat cggcactgcc 2100 tcacgcccta gcgaggtgtg tatggtggac ggaacggaca tgtgtttagc tgattttcat 2160 gctggcattt tcctgaaagg acaggaacat gctgtgttcg cctgtgtcac ctccaacggg 2220 tggtacgcga ttgatgacga ggacttttac ccttggacac cggacccgtc cgacgttctg 2280 gtgtttgtcc cgtacgatca agaaccgctt aacggagagt ggaaaacaaa ggtccagaaa 2340 aggctcaagg gagccgggca atccagcccg gcaaccgggt cacagaacca atcaggcaac 2400 actgggagca tcatcaacaa ttactacatg cagcagtacc aaaactccat ggacacacaa 2460 cttggtgaca acgctacaag cggaggctca aacgaggggt ccacggacac aacctccacc 2520 cac acaacca acactcagaa caacgactgg ttctcgaagc tggccagttc cgctttcagc 2580 ggtcttttcg gcgctcttct cgccgacaag aaaaccgagg agaccactct tctcgaggac 2640 cgcatcctca ctactcgtaa cggacacacc acctcgacaa ccccagtcgag cgtt ggagtc 2700 acgtacgggt atgcaacagc tgaggatttc gtgagcgggc caaacacctc tggtctcgag 2760 accagggttg cccaggcaga gcggttcttt aaaacccacc tgttcgactg ggtcaccagt 2820 gacccattcg gacggtgcca cctgctggaa cttccaactg accacaaagg tgtctacggc 2880 agcctgaccg actcgtatgc ttatatgagg aacggctggg atgttgaagt cactgcagg 2940 ggaaaccagt tcaatggagg atgcctgttg gtggccatgg tgccagaact ttgctccata 3000 cagaagaggg agctgtacca gctcacgctc tttcctcacc agttcatcaa ccctcggacg 3060 aacatgacag cacacatcac tgtgcccttt gttggcgtca accgttat ga ccagtacaag 3120 gtacacaaac cttggaccct cgtggttatg gttgtagccc ccctgactgt caacagtgaa 3180 ggtgccccgc aaatcaaggt gtatgccaac atcgcaccta ccaacgtaca cgtcgcgggt 3240 gagttccctt ccaaagaggg gatcttccct gtggcttgca gcgatggtta tggcggtctg 3300 gtgaccactg acccgaaaac ggctgacccc gcttacggga aagtgtttaa ccccccccgc 3360 aacatgtt gc cggggcggtt caccaatttt cttgacgtgg ctgaggcgtg ccccacgttt 3420 ctccacttcg agggtgacgt gccatacgtg accacgaaga cggattcaga cagggtgctc 3480 gctcagttcg acttgtcttt ggcagcaaag cacatgtcga acaccttcct tgcaggtctc 3600 actgacgcga aggcgcgtta catgattgcg tatgctcctc ctggcatgga accacctaaa 3660 acgccagagg cggctgccca ctgcattcat gctgaatggg acacagggtt gaactcaaaa 3720 ttcacatttt caatccctta cctttcggcg gctgattacg cttacacagc gtctgacact 3780 gctgagacca caaatgtaca g ggatgggtt tgcctgtttc aaataacaca cgggaaagct 3840 gacggcgacg cactggtcgt tttggctagc gccggaaagg actttgagct gcgcctgccg 3900 gtggatgctc gcacacagac tacctccgcg ggcgagtcag ctgaccccgt gaccgccacc 3960 gttgagaatt acggtggcga gacacaggtc cagaggcgcc aacacacgga cgtctcattt 4020 atattagaca gatttgtgac agtgacacca aaagaccaaa cac caaccca acagcttacc acaaggcacc actcacccga 4260 cttgcactgc cttacacggc gccacaccgc gtgttggcta ctgtttacaa cgggaactgc 4320 aagtatggtg acggcacggt ggccaatgtg agaggtgacc tgcaagtgtt ggcccagaag 4380 gcggcgagag cg ctgcctac ctccttcaac tacggtgcca ttaaagctac tcgggtgact 4440 gaactgcttt accgcatgaa gagggctgag acatactgcc cccggcctct tttggccatt 4500 cacccggacc aggctagaca caagcagaag attgtggcac cggtggaaca gcttctaaat 4560 tttgacctgc tcaaattggc gggagatgtg gagtccaacc ctgggccctt cttcttctcc 4620 gacgtcaggt caaatttctc aaaactggta ga aaccatca atcagatgca ggaggacatg 4680 tcaacaaaac acgggcctga ctttaaccgg ttggtgtccg catttgagga attggccact 4740 ggaggtgaagg ctatcagggc cggtctcgac gaggccaaac cctggtacaa actcatcaag 4800 ctcctgagcc gcttgtcgtg catggcc gct gtagcagcac ggtcaaagga cccagtcctt 4860 gtggccatca tgctggctga caccggtctt gagattctgg acagcacctt tgtcgtgaag 4920 aagatctccg actcgctctc cagtctcttt cacgtgccgg cccccgtctt cagtttcgga 4980 gccccgattc tgttggccgg gttggtcaaa gtcgcctcga gtttcttccg gtccacaccc 5040 gaagaccttg agagagcaga aaaacagctc aaagcac gtg acattaacga catattcgcc 5100 attctcaaga acggcgagtg gctggtcaag ctgatccttg ccatccgcga ctggatcaaa 5160 gcgtggatcg cctcagaaga aaagtttgtc accatgacgg acttggtgcc tggtatcctt 5220 gaaaagcagc gggatctcaa cgacccga gt aagtacaagg aagccaagga gtggctcgac 5280 aacgcgcgcc aggcgtgttt gaagagcggg aacgttcaca ttgccaattt gtgcaaagtg 5340 gtcgccccgg cacccagcaa gtcgagaccc gaacccgtgg tcgtttgcct ccgcggcaaa 5400 tccggccagg ggaagagttt ccttgcgaac gtgctcgcgc aagcaatctc cacccacttc 5460 accggcagaa ctgattcggt ttggtactgc ccgcctgacc ctgaccactt cgacggttac 5520 aaccagcaga ccgttgtcgt gatggacgat ttgggccaga accccgatgg caaggacttc 5580 aagtacttcg cccagatggt ttcgaccacg gggttcatcc cgcccatggc ctcgcttgag 5640 gacaaaggca agcctttcaa cagcaa agtc atcattgcta ccaccaacct gtactcgggt 5700 ttcaccccga gaacaatggt gtgtcctgac gcgctgaacc ggaggttcca ctttgacatc 5760 gacgtgagtg ccaaggacgg gtacaaagtt aacaacaaat tggacataat caaagctctt 5820 gaagacaccc acaccaaccc agtggcgatg ttccaatacg actgtgccct tctaaacggt 5880 atggcagttg aaatgaagag aatgcaacag gatatgttca agcctcaacc acccctccag 5940 a acgtgtacc aactcgttca cgaggtgatt gaacgggtcg agctccacga gaaggtgtcg 6000 agccacccga ttttcaaaca gatatcaatt ccttcccaaa agtctgtgtt gtacttcctc 6060 attgagaaag gccaacacga agcagcaatt gaattctttg agggaatggt gcatgactcc 6120 atcaaggaag agctccggcc cctcatccaa cagacctcat ttgtgaaacg cgcttttaag 6180 cgcctgaagg aaaactttga gactgttgcc ctgtgtttga ctcttttggc aaacatagtg 6240 atcatgatcc gcgagactcg caagagacaa cagatggtgg acgatgcagt gaatgactac 6300 attgagaagg caaacatcac cacagatgac aagactcttg acgaggcgga aaagaaccct 6360 ctagagacca gc ggtgccag cactattggt ttcagagaga gaactctccc ggggcacaag 6420 gcgagcgatg acgtgagctc cgagcccgcc aaacccgt gg aggaccgacc acaagctgaa 6480 gggccctacg ccggaccact tgagcgtcag aaacctctga gagtgaaaac caagttgcca 654 0 caacaggagg gaccctacgc tggcccgatg gatagacaga aaccgttgaa agtgagagca 6600 agagccccgg tcgtgaagga gggaccctac gagggaccgg tgaagaagcc tgtcgctttg 6660 aaagtgaaag ccaagaactt gattgtcact gaaagtggtg ccccaccgac cgacttgcag 6720 aagatggtca tgggcaacac taagcctgtt gagctcatcc tcgacgggaa gacggtagcc 6780 atctgctgtg ctaccggag t gtttggcact gcctacctcg tacctcgtca cctcttcgcg 6840 gagaagtacg acaagataat gttggacggt agagccatga cagacagtga ctacagagtg 6900 tttgagtttg agattaaagt aaaaggacag gacatgctct cagacgctgc actcatggtg 6960 cttca ccgtg ggaaccgcgt gagagacatc acgaaacatt ttcgtgacac agcaagaatg 7020 aagaaaggca cccccgttgt cggtgtgatc aacaacgccg acgttgggag actgattttc 7080 tctggagagg cccttaccta caaagacatt gtagtgacca tggatggaga caccatgccg 7140 ggcctgtttg cctacagagc cgccaccaag gctggttact gcgggggagc cgttctcgcc 7200 aaggacggag ccgacacatt catcgttggc acccact ccg caggtggtaa cggagttgga 7260 tactgctcgt gcgtgtccag gtccatgctc ctgaaaatga aggcacacat tgaccctgaa 7320 ccacaccacg aggggttgat tgttgatacc agagatgtgg aagagcgcgt gcatgtcatg 7380 cgtaaaacca agcttgcacc caccgtggca cacggtgtgt ttaaccctga atttggtccc 7440 gctgccttgt ccaacaagga cccgcggctg aacgaagggg ttgtcctcga tgaagtcatc 7500 ttctccaaac acaagggaga cacgaaaatg tctgaggagg acaaagcgct gttccgccgc 7560 tgcgctgccg actacgcgtc gcacttgcac agcgtgctgg ggacggcaaa tgccccattg 7620 agcatctatg aggccatcaa aggc gtcgac gggctcgatg ccatggagcc ggacaccgcg 7680 cccggcctcc cctgggccct ccaggggaaa cgccgtggtg cgttgattga cttcgagaac 7740 ggcacggtcg gacccgaagt cgaggctgcc ctaaagctca tggagaaaag agagtacaaa 7800 tttgctt gcc agaccttcct gaaagacgag attcgtccga tggaaaaagt acgtgctggc 7860 aagactcgca ttgtcgacgt tttgcccgtg gaacacattc tttacaccag gatgatgatt aaccactgca gtgacgcaat gaacatcatg 8100 tttgaggagg tatttcgcac agacttcggt ttccacccaa atgctgagtg gattctgaag 8160 actcttgtga acacggagca cgcctatgag aacaaacgta tcactgttga gggcgggatg 8220 ccgtctggct gttccgcgac aagcat catc aacacaattt tgaacaacat ttatgtgctc 8280 tacgctcttc gtagacacta tgagggagtt gagctggaca cctacaccat gatctcctac 8340 ggagatgaca tcgtggttgc aagtgactac gatctggatt ttgaggctct caaaccccac 8400 ttcaaatctc ttggtcaaac catcactcca gctgacaaaa gcgacaaagg ttttgttctt 8460 ggtcactcca ttaccgatgt cactttcctc aaaagacact t ccacatgga ctatggaact 8520 gggttttaca aacctgtgat ggcctcaaag accctcgagg ccattctctc ctttgcacgc 8580 cgtgggacca tacaggagaa gttgatctcc gtggcaggac tcgccgtcca ctcaggacct 8640 gacgagtacc ggcgtctctt tgagccctt c cagggtctct tcgagattcc aagctacaga 8700 tcactttacc tgcgttgggt gaacgccgtg tgcggtgacg cataatccct cagatgtcac 8760 aattggcaga aagactctga ggcgagcgac gccgtaggag tgaaaagccc gaaagggctt 8820 ttcccgcttc ctattccaa aaaaaaaaaa aaaactagtt ctagagcggc cgccaccgcg 8880 gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgct tgg cgtaatcatg 8940 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 9000 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 9060 gttgcgctca ctgcccgctt tccagtcggg aa acctgtcg tgccagctgc attaatgaat 9120 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 9180 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 9240 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 9300 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccg ccc 9360 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 9420 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 9480 gccgcttacc ggatacctgt ccgcctttct cccttcgg ga agcgtggcgc tttctcatag 9540 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 9600 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 9660 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 9720 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 9780 aaggacagta tttggta tct gcgctctgct gaagccagtt accttcggaa aaagagttgg 9840 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 9900 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacgggg tc 9960 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 10020 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 10080 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 10140 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 10200 ggagggctta ccatctggcc ccagtgct gc aatgataccg cgagaccccac gctcaccggc 10260 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 10320 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 10380 gccagttaat agttt gcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 10440 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 10500 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 10560 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 10620 gccatccgta agatgctttt ctgt gactgg tgagtactca accaagtcat tctgagaata 10680 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 10740 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 10800 gatcttaccg ctgt tgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 10860 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 10920 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 10980 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 11040 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 11090 <210> 3 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> O1manisa_VP1 <400> 3 actacctccg cgggcgagtc agctgacccc gtgaccgcca ccgttgagaa ttacggtggc 60 gagacacagg tccagaggcg ccaacacacg gacgtctcat ttatattaga cagatttgtg 120 acagtgacac caaaagacca aattaatgta ttggacctga tgcaaacccc tgctcacact 180 t tggtgggag cactccttcg tactgccact tactatttcg ctgacttaga ggtggcagtg 240 aagcacaagg gaaacctcac ctgggtcccg aacggggcgc ctgaagcggc gttggacaac 300 accaccaacc caacagctta ccacaaggca ccactcaccc gacttgcact gccttacacg 360 gcgccacacc gcgtgttggc tactgtttac aacgggaact gcaagtatgg tgacggcacg 420 gtggccaatg tgagaggtga cctgcaagtg ttggcccaga aggcggcgag agcgctgcct 480 acctccttca actacggtgc cattaaagct actcgggtga ctgaactgct ttaccgcatg 540 aagagggctg agacatactg cccccggcct cttttggcca ttcacccgga ccaggctaga 600 cacaagcaga agattgt ggc accggtggaa cag 633 <210> 4 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> O JC_VP1 < c ccccacact 180 ctagtagggg cgctcctccg cgctgccact tactatttcg ctgacctaga ggtggcagg 240 aaacacgagg gggaccttac ctgggtgcca aacggagcac ctgaagcagc cttggacaac 300 accaccaacc caacggcgta ccataaggcg ccgcttaccc ggcttgcat t gccctacacg 360 gcaccacacc gtgttttggc caccgtttac aacgggaact gcaaatacac cgggggctca 420 ctgcccaacg tgagaggcga tctccaagtg ctggcaccga aagcggcgag gccattgccc 480 acttctttca actacggtgc tatcaaagcc actcgggtaa cagaactgct gtaccgcatg 540 aagagggccg agacgtactg ccctcggccc ctcttggctg t ccacccgag tgcggctaga 600 cacaaacaga aaatagtggc acctgtaaag cag 633 <210> 5 <211> 633 <212> DNA <213> Artificial Sequence <220> <223 > PA2_VP1 <400> 5 accacctcca caggtgagtc agctgacccc gtgactgcca ctgttgagaa ctacggtggc 60 gagacgcagg tccagagacg ccagcacacg gacgtctcgt tcatactgga cagatttgtg 120 aaagtgacac caaaagacca aattaatgtg ttgg acctga tgcagacccc cgcccacact 180 ttggtaggtg cgcttctccg caccgccacc tactacttcg cagacctaga ggtggcagtg 240 aaacacgagg ggaaccttac ctgggtcccg aatggggcgc ccgagacagc gttggataac 300 accaccaatc caacggctta ccacaagg ca cctctcaccc ggcttgcgct gccttacacg 360 gcaccacacc gtgtcttggc tactgtttac aacgggaact gcaagtatgg cgagagctcc 420 acaaccaacg tgagaggtga cctgcaagtg ttggcccaga aagcggcgag agcgctgcct 480 acctccttta actacggtgc cattaaggcc actcgggtga ctgaactgct ttaccgcatg 540 aagagggctg aaacatatactg cccccggcct cttttggcta ttcacccgag cgaagcaaga 600 cacaaacaaa agatagtggc acctgtgaaa cag 633 <210> 6 <211> 11100 <212> DNA <213> Artificial Sequence <220 > <223> O JC-VP1 Recombianant plasmid <400> 6 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agt gttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt ttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 ccc ccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggct gcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcact a tagggttgaa aggggggcgct 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 aacg ctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 cccccccccc ccccctaggt tttaccgtcg ttcccgacgt taatggggaa acaaccaa 1 080 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 c ccgcctggt ctttccaggt ctagaggggc gacattttgt actgtgcttg 1380 actccacgct cggtccacta gcgagtgtta gtagtagcac tgttgcttcg tagcggagca 1440 tgatggccgt gggagcttcc ccttggtaac aaggacccac ggggccaaaa gccacgtcct 1500 accg gaccca 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 actgttt tcatgaatac a actgactgt ttcaccgccc tgttacacgc tctcagagag 1800 atcaaaacac tgtttctttt acggacacaa ggaaagatgg aattcacact ttacaacggt 1860 gagaagaaaa ccttctactc cagacccaac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggta tgt 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 agcccggcaa ccgggtcaca gaaccaatca 2400 ggcaacactg ggagcatcat caacaattac tacatgcagc agtaccaaaa ctccatggac 2460 acacaacttg gtgacaacgc tacaagcgga ggctcaaacg aggggtccac ggacacaacc 2520 tccacccaca caaccaacac tcagaacaac gactggttct cgaagctggc cagttccgct 2580 ttcagcggtc tttcggcgc tcttctcgcc gacaagaaaa ccgaggagac cactcttctc 2640 gaggaccgca tcctcactac tcgtaacgga cacaccacct cgacaaccca gtcgagcgtt 2700 ggaggtcacgt acgggtatgc aacagctgag gatttcgtga gcgggccaaa cacctctggt 2760 ctcgagacca gggttgccca ggcaga gcgg ttctttaaaa cccacctgtt cgactgggtc 2820 accagtgacc cgttcggacg gtgccacctg ctggaacttc caactgacca caaaggtgtc 2880 tacggcagcc tgaccgactc gtatgcttat atgaggaacg gctgggatgt tgaagtcact 2940 gcagtgggaa accagttcaa tggaggatgc ctgttggtgg ccatggtgcc a tag cccccct gaccgtcaac 3180 agtgaaggtg ccccgcaaat caaggtgtat gccaacatcg cacctaccaa cgtacacgtc 3240 gcgggtgagt tcccttccaa agaggggatc ttccctgtgg cttgcagcga tggttatggc 3300 ggtctggtga ccactgaccc gaaaacggct gaccccgctt acgggaaagt gtttaacccc 3360 ccccgcaaca tgttgccggg gcggttcacc aattttcttg acgtggctga ggcgt gcccc 3420 acgtttctcc acttcgaggg tgacgtgcca tacgtgacca cgaagacgga ttcagacagg 3480 gtgctcgctc agttcgactt gtctttggca gcaaagcaca tgtcgaacac cttccttgca 3540 ggtctcgccc agtactacac acagtacagc gg cac catca acctgcactt catgttcaca 3600 gggcctactg acgcgaaggc gcgttacatg attgcgtatg ctcctcctgg catggaacca 3660 cctaaaacgc cagaggcggc tgcccactgc attcatgctg aatgggacac agggttgaac 3720 tcaaaattca cattttcaat cccttacctt tcggcggctg attacgctta cacagcgtct 3780 gacactgctg agaccacaaa tgtacaggga tgggtttgcc tgtttcaaat aacacacggg 3840 aa agctgacg gcgacgcact ggtcgttttg gctagcgccg gaaaggactt tgagctgcgc 3900 ctgccggtgg atgctcgcac acagaccact tcgacaggcg agtcggctga ccccgtgact 3960 gccaccgttg agaactacgg tggcgagaca cagatccaga ggcgccacca cacagacgtc 4020 tcgttcatac tggacagatt tgtgaaagtc acaccaaaag actcaacaaa tgtactggac 4080 ctgatgcaga ccccccccca cactctagta ggggcgctcc tccgcgctgc cacttactat 4140 ttcgctgacc tagaggtggc agtgaaacac gagggggacc ttacctgggt gccaaacgga 4200 gcacctgaag cagccttgga caacaccacc aacccaacgg cgtaccataa ggcgccgctt 4260 acccggcttg cattgc ccta cacggcacca caccgtgttt tggccaccgt ttacaacggg 4320 aactgcaaat acaccggggg ctcactgccc aacgtgagag gcgatctcca agtgctggca 4380 ccgaaagcgg cgaggccatt gcccacttct ttcaactacg gtgctatcaa agccactcgg 4440 gtaacaga ac tgctgtaccg catgaagagg gccgagacgt actgccctcg gcccctcttg 4500 gctgtccacc cgagtgcggc tagacacaaa cagaaaatag tggcacctgt aaagcagctt 4560 ctaaattttg acctgctcaa attggcggga gatgtggagt ccaaccctgg gcccttcttc 4620 ttctccgacg tcaggtcaaa tttctcaaaa ctggtagaaa ccatcaatca gatgcaggag 4680 gacatgtcaa caaaacac gg gcctgacttt aaccggttgg tgtccgcatt tgaggaattg 4740 gccactggag tgaaggctat cagggccggt ctcgacgagg ccaaaccctg gtacaaactc 4800 atcaagctcc tgagccgctt gtcgtgcatg gccgctgtag cagcacggtc aaaggaccca 4860 gt ccttgtgg ccatcatgct ggctgacacc ggtcttgaga ttctggacag cacctttgtc 4920 gtgaagaaga tctccgactc gctctccagt ctctttcacg tgccggcccc cgtcttcagt 4980 ttcggagccc cgattctgtt ggccgggttg gtcaaagtcg cctcgagttt cttccggtcc 5040 acacccgaag accttgagag agcagaaaaa cagctcaaag cacgtgacat taacgacata 5100 ttcgccattc tcaagaacgg cgagtggctg gtcaag c gcgccaggc gtgtttgaag agcgggaacg ttcacattgc caatttgtgc 5340 aaagtggtcg ccccggcacc cagcaagtcg agacccgaac ccgtggtcgt ttgcctccgc 5400 ggcaaatccg gccaggggaa gagtttcctt gcgaacgtgc tcgcgcaagc aatctccacc 5460 cacttcaccg gcagaactga ttcggtttgg tactgcccgc ctgaccctga ccacttcgac 5520 ggttacaacc agcagaccgt tgtcgtgatg gacg atttgg gccagaaccc cgatggcaag 5580 gacttcaagt acttcgccca gatggtttcg accacggggt tcatcccgcc catggcctcg 5640 cttgaggaca aaggcaagcc tttcaacagc aaagtcatca ttgctaccac caacctgtac 5700 tcgggtttca ccccgagaac aat ggtgtgt cctgacgcgc tgaaccggag gttccacttt 5760 gacatcgacg tgagtgccaa ggacgggtac aaagttaaca acaaattgga cataatcaaa 5820 gctcttgaag acacccacac caacccagtg gcgatgttcc aatacgactg tgcccttcta 5880 aacggtatgg cagttgaaat gaagagaatg caacaggata tgttcaagcc tcaaccaccc 5940 ctccagaacg tgtaccaact cgttcacgag gtgattgaac gggtcgagct ccac gagaag 6000 gtgtcgagcc acccgatttt caaacagata tcaattcctt cccaaaagtc tgtgttgtac 6060 ttcctcattg agaaaggcca acacgaagca gcaattgaat tctttgaggg aatggtgcat 6120 gactccatca aggaagagct ccggcccctc atccaacaga cct catttgt gaaacgcgct 6180 tttaagcgcc tgaaggaaaa ctttgagact gttgccctgt gtttgactct tttggcaaac 6240 atagtgatca tgatccgcga gactcgcaag agacaacaga tggtggacga tgcagtgaat 6300 gactacattg agaaggcaaa catcaccaca gatgacaaga ctcttgacga ggcggaaaag 6360 aaccctctag agaccagcgg tgccagcact attggtttca gagagagaac tctcccgggg 6420 cacaaggcga gcgatgacgt gagctccgag cccgccaaac ccgtggagga ccgaccacaa 6480 gctgaaggac cttacgaggg accggtgaag aagcctgtcg ctttgaaagt gaagctaag 6540 aacttgattg tcactgaggg gccatatgaa ggaccagtga agaaacctgt cgcttt gaaa 6600 gtgaaagcaa aagccccgat tgtcactgaa ggaccctacg agggaccggt gaagaagcct 6660 gtcgctttga aagtgaaagc caagaacttg attgtcactg agagtggtgc cccaccgacc 6720 gacttgcaga agatggtcat gggcaacact aagcctgttg agctcatcct cgacgggaag 6780 acggtagcca tctgctgtgc taccggagtg tttggcactg cctacctcgt acctcgtcac 6840 ctcttcgc gg agaagtacga caagataatg ttggacggta gagccatgac agacagtgac 6900 tacagagtgt ttgagtttga gattaaagta aaaggacagg acatgctctc agacgctgca 6960 ctcatggtgc ttcaccgtgg gaaccgcgtg agagacatca cgaaacattt tcgtgacaca 70 20 gcaagaatga agaaaggcac ccccgttgtc ggtgtgatca acaacgccga cgttgggaga 7080 ctgattttct ctggagaggc ccttacctac aaagacattg tagtgaccat ggatggagac 7140 accatgccgg gcctgtttgc ctacagagcc gccaccaagg ctggttactg cgggggagcc 7200 gttctcgcca aggacggagc cgacacattc atcgttggca cccactccgc aggtggtaac 7260 ggagttggat actgctcgtg cgtgt ccagg tccatgctcc tgaaaatgaa ggcacacatt 7320 gaccctgaac cacaccacga ggggttgatt gttgatacca gagatgtgga agagcgcgtg 7380 catgtcatgc gtaaaaccaa gcttgcaccc accgtggcac acggtgtgtt taaccctgaa 7440 tttggtcccg ctgccttgtc caacaaggac ccgcggctga acgaaggggt tgtcctcgat 7500 gaagtcatct tctccaaaca caagggagac acgaaaatgt ctgaggagga caaagcgctg 7560 ttccgccgct gcgctgccga ctacgcgtcg cacttgcaca gcgtgctggg gacggcaaat 7620 gccccattga gcatctatga ggccatcaaa ggcgtcgacg ggctcgatgc catggagccg 7680 gacaccgcgc ccggcctcc c ctgggccctc caggggaaac gccgtggtgc gttgattgac 7740 ttcgagaacg gcacggtcgg acccgaagtc gaggctgccc taaagctcat ggagaaaaga 7800 gagtacaaat ttgcttgcca gaccttcctg aaagacgaga ttcgtccgat ggaaaaagta 78 60 cgtgctggca agactcgcat tgtcgacgtt ttgcccgtgg aacacatct ttacaccagg 7920 atgatgattg gcagattctg tgctcaaatg cacacaaaca atggaccgca gattggctca 7980 gcggtcggtt gcaatcctga tgttgattgg caaagatttg gcacacattt tgctcagtac 8040 agaaacgtgt gggatgtgga ctattcggcc tttgatgcta accactgcag tgacgcaatg 8100 aacatcatgt ttgaggaggt atttcgcaca gact tcggtt tccacccaaa tgctgagtgg 8160 attctgaaga ctcttgtgaa cacggagcac gcctatgaga acaaacgtat cactgttgag 8220 ggcgggatgc cgtctggctg ttccgcgaca agcatcatca acacaatttt gaacaacatt 8280 tatgtgctct acgctcttcg tagac actat gagggagttg agctggacac ctacaccatg 8340 atctcctacg gagatgacat cgtggttgca agtgactacg atctggattt tgaggctctc 8400 aaaccccact tcaaatctct tggtcaaacc atcactccag ctgacaaaag cgacaaaggt 8460 tttgttcttg gtcactccat taccgatgtc actttcctca aaagacactt ccacatggac 8520 tatggaactg ggttttacaa acctgtgatg gcctcaaaga ccctcga ggc cattctctcc 8580 tttgcacgcc gtgggaccat acaggagaag ttgatctccg tggcaggact cgccgtccac 8640 tcaggacctg acgagtaccg gcgtctcttt gagcccttcc agggtctctt cgagattcca 8700 agctacagat cactttacct gcgttgggtg aacgccgt gt gcggtgacgc ataatccctc 8760 agatgtcaca attggcagaa agactctgag gcgagcgacg ccgtaggagt gaaaagcccg 8820 aaagggcttt tcccgcttcc tattccaaaa aaaaaaaaaa aaaactagtt ctagagcggc 8880 cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg 8940 cgtaatcatg gtcatagctg tttcctgtgt gaaattg tta tccgctcaca attccacaca 9000 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 9060 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 9120 attaatgaat cggccaacgc gc ggggagag gcggtttgcg tattgggcgc tcttccgctt 9180 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 9240 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 9300 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 9360 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tgg cgaaacc 9420 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 9480 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 9540 tttctcatag ctcacgctgt aggtatctca gt tcggtgta ggtcgttcgc tccaagctgg 9600 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 9660 ttgagtccaa cccggtaaga aa agagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg 9900 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt 9960 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agg gattttg gtcatgagat 10020 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct 10080 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta 10140 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa 10200 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac 10260 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa 10320 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag 10380 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 10440 tgtc acgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag 10500 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg 10560 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc 10620 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat 10680 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata 10740 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa 10800 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca 10860 actgatctt c agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc 10920 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc 10980 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg 11040 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 11100 11100 <210> 7 <211> 11 097 <212> DNA <213> Artificial Sequence <220> <223> O PA2-VP1 Recombianant plasmid <400> 7 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 atttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtgg actc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcg agaaagg 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 cacg acgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa aggggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagt tggact 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 1 020 cccccccccc ccccctaggt tttaccgtcg ttcccgacgt taatgggggaa acaaccacaa 1080 gcttaacacc gtcttgcccg acgtaaaagg gctgcaacca aaaagcttgt gccgcctttc 1140 ccggcgttaa tgggaggtaa ccacaagaca aaccttcacc cggaagtaa a 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 cgc gacactc 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 cagacc caac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggtatgt tgatgagcct ttctttgact gggtctacga ctcgcctgaa 1980 aacctcactc ttgaggcaat caaacagttg gaagagacaa ccggtcttga gctgcacgag 2040 ggtgggaccac 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 cga tcaagaa ccgcttaacg gagagtgggaa aacaaaggtc 2340 cagaaaaggc tcaagggagc cgggcaatcc agcccggcaa ccgggtcaca gaaccaatca 2400 ggcaacactg ggagcatcat caacaattac tacatgcagc agtaccaaaa ctccatggac 2460 acacaacttg gtgacaacg c tacaagcgga ggctcaaacg aggggtccac ggacacaacc 2520 tccacccaca caaccaacac tcagaacaac gactggttct cgaagctggc cagttccgct 2580 ttcagcggtc ttttcggcgc tcttctcgcc gacaagaaaa ccgaggagac cactcttctc 2640 gaggaccgca tcctcactac tcgtaacgga cacaccacct cgacaaccca gtcgagcgtt 2700 ggaggtcacgt acgggtatgc aacagctgag gatttc gtga gcgggccaaa cacctctggt 2760 ctcgagacca gggttgccca ggcagagcgg ttctttaaaa cccacctgtt cgactgggtc 2820 accagtgacc cgttcggacg gtgccacctg ctggaacttc caactgacca caaaggtgtc 2880 tacggcagcc tgaccgact c gtatgcttat atgaggaacg gctgggatgt tgaagtcact 2940 gcagtgggaa accagttcaa tggaggatgc ctgttggtgg ccatggtgcc agaactttgc 3000 tccatacaga agagggagct gtaccagctc acgctctttc ctcaccagtt catcaaccct 3060 cggacgaaca tgacagcaca catcactgtg ccctttgttg gcgtcaaccg ttatgaccag 3120 tacaaggtac acaaaccttg gaccctcgtg gttatggttg tagcccccct gaccgtcaac 3180 agtgaaggtg ccccgcaaat caaggtgtat gccaacatcg cacctaccaa cgtacacgtc 3240 gcgggtgagt tcccttccaa agaggggatc ttccctgtgg cttgcagcga tggttatggc 3300 ggtctggtga ccactgaccc gaaaacggct gaccccgctt ac gggaaagt gtttaacccc 3360 ccccgcaaca tgttgccggg gcggttcacc aattttcttg acgtggctga ggcgtgcccc 3420 acgtttctcc acttcgaggg tgacgtgcca tacgtgacca cgaagacgga ttcagacagg 3480 gtgctcgctc agttcgactt gtctttggca gcaaagcaca tgtcgaacac cttccttgca 3540 ggtctcgccc agtactacac acagtacagc ggcaccatca acctgcactt catgttcaca 3600 gggcctactg acgcgaaggc gcgttacatg attgcgtatg ctcctcctgg catggaacca 3660 cctaaaacgc cagaggcggc tgcccactgc attcatgctg aatgggacac agggttgaac 3720 tcaaaattca catttcaat cccttacctt tcggcggctg attac gctta cacagcgtct 3780 gacactgctg agaccacaaa tgtacaggga tgggtttgcc tgtttcaaat aacacacggg 3840 aaagctgacg gcgacgcact ggtcgttttg gctagcgccg gaaaggactt tgagctgcgc 3900 ctgccggtgg atgctcgcac acagaccacc tccacaggtg agtcagctga ccccgtgact 3960 gccactgttg agaactacgg tggcgagacg caggtccaga gacgccagca cacggacgtc 4020 tcgttcatac tggacagatt tgtgaaagtg acaccaaaag accaaattaa tgtgttggac 4080 ctgatgcaga cccccgccca cactttggta ggtgcgcttc tccgcaccgc cacctactac 4140 ttcgcagacc tagaggtggc agtgaaacac gaggggaacc ttacctgggt c ccgaatggg 4200 gcgcccgaga cagcgttgga taacaccacc aatccaacgg cttaccacaa ggcacctctc 4260 acccggcttg cgctgcctta cacggcacca cat gaagagg gctgaaacat actgcccccg gcctcttttg 4500 gctattcacc cgagcgaagc aagacacaaa caaaagatag tggcacctgt gaaacagctt 4560 ctaaattttg acctgctcaa attggcggga gatgtggagt ccaaccctgg gcccttcttc 4620 ttctccgacg tcaggtcaaa tttctcaaaa ctggtagaaa ccatcaatca gatgcaggag 4680 gacatgtcaa caaaacacgg gcctgacttt aaccggttgg tgtccgcatt tgaggaattg 4740 gccactggag tgaaggctat cagggccggt ctcgacgagg ccaaaccctg gtacaaactc 4800 atcaagctcc tgagccgctt gtcgtgcatg gccgctgtag cagcacggtc aaaggaccca 4860 gtccttgtgg ccatcatgct ggctga cacc ggtcttgaga ttctggacag cacctttgtc 4920 gtgaagaaga tctccgactc gctctccagt ctctttcacg tgccggcccc cgtcttcagt 4980 ttcggagccc cgattctgtt ggccgggttg gtcaaagtcg cctcgagttt cttccggtcc 5040 acaccc gaag accttgagag agcagaaaaa cagctcaaag cacgtgacat taacgacata 5100 ttcgccattc tcaagaacgg cgagtggctg gtcaagctga tccttgccat ccgcgactgg 5160 atcaaagcgt ggatcgcctc agaagaaaag tttgtcacca tgacggactt ggtgcctggt 5220 atccttgaaa agcagcggga tctcaacgac ccgagtaagt acaaggaagc caaggagtgg 5280 ctcgacaacg cgcgccaggc gt gtttgaag agcgggaacg ttcacattgc caatttgtgc 5340 aaagtggtcg ccccggcacc cagcaagtcg agacccgaac ccgtggtcgt ttgcctccgc 5400 ggcaaatccg gccaggggaa gagtttcctt gcgaacgtgc tcgcgcaagc aatctccacc 5460 cacttcaccg gcagaactga ttcggtttgg tactgccc gc ctgaccctga ccacttcgac 5520 ggttacaacc agcagaccgt tgtcgtgatg gacgatttgg gccagaaccc cgatggcaag 5580 gacttcaagt acttcgccca gatggtttcg accacggggt tcatcccgcc catggcctcg 5640 cttgaggaca aaggcaagcc tttcaac agc aaagtcatca ttgctaccac caacctgtac 5700 tcgggtttca ccccgagaac aatggtgtgt cctgacgcgc tgaaccggag gttccacttt 5760 gacatcgacg tgagtgccaa ggacgggtac aaagttaaca acaaattgga cataatcaaa 5820 gctcttgaag acacccacac caacccagtg gcgatgttcc aatacgactg tgcccttcta 5880 aacggtatgg cagttgaaat gaagagaatg caacaggata tgttcaagcc tcaaccaccc 594 0 ctccagaacg tgtaccaact cgttcacgag gtgattgaac gggtcgagct ccacgagaag 6000 gtgtcgagcc acccgatttt caaacagata tcaattcctt cccaaaagtc tgtgttgtac 6060 ttcctcattg agaaaggcca acacgaagca gcaattgaat tctttgaggg aatggtgcat 6120 gactccatca aggaagagct ccggcccctc atccaacaga cctcatttgt gaaacgcgct 6180 tttaagcgcc tgaaggaaaa ctttgagact gttgccctgt gtttgactct tttggcaaac 6240 atagtgatca tgatccgcga gactcgcaag agacaacaga tggtggacga tgcagtgaat 6300 gactacattg agaaggcaaa catcaccaca gatgacaaga ctcttgacga ggcggaaaag 6360 aaccc tctag agaccagcgg tgccagcact attggtttca gagagagaac tctcccgggg 6420 cacaaggcga gcgatgacgt gagctccgag cccgccaaac ccgtggagga ccgaccacaa 6480 gctgaagggc cctacgccgg accacttgag cgtcagaaac ctctgagagt gaaaaccaag 654 0 ttgccacaac aggaggggacc ctacgctggc ccgatggata gacagaaacc gttgaaagtg 6600 agagcaagag ccccggtcgt gaaggaggga ccctacgagg gaccggtgaa gaagcctgtc 6660 gctttgaaag tgaaagccaa gaacttgatt gtcactgaaa gtggtgcccc accgaccgac 6720 ttgcagaaga tggtcatggg caacactaag cctgttgagc tcatcctcga cgggaagacg 6780 gtagccatct gct gtgctac cggagtgttt ggcactgcct acctcgtacc tcgtcacctc 6840 ttcgcggaga agtacgacaa gataatgttg gacggtagag ccatgacaga cagtgactac 6900 agagtgtttg agtttgagat taaagtaaaa ggacaggaca tgctctcaga cgctgcactc 6960 atggtgcttc accgtgggaa ccgcgtgaga gacatcacga aacattttcg tgacacagca 7020 agaatgaaga aaggcacccc cgttgtcggt gtgatcaaca acgccgacgt tgggagactg 7080 attttctctg gagaggccct tacctacaaa gacattgtag tgaccatgga tggagacacc 7140 atgccgggcc tgtttgccta cagagccgcc accaaggctg gttatgcgg gggagccgtt 7200 ctcgccaagg acggagccga cacattcatc gt tggcaccc actccgcagg tggtaacgga 7260 gttggatact gctcgtgcgt gtccaggtcc atgctcctga aaatgaaggc acacattgac 7320 cctgaaccac accacgaggg gttgattgtt gataccagag atgtggaaga gcgcgtgcat 7380 gtcatgcgta aaaccaagct tgcaccca cc gtggcacacg gtgtgtttaa ccctgaattt 7440 ggtcccgctg ccttgtccaa caaggacccg cggctgaacg aaggggttgt cctcgatgaa 7500 gtcatcttct ccaaacacaa gggagacacg aaaatgtctg aggaggacaa agcgctgttc 7560 cgccgctgcg ctgccgacta cgcgtcgcac ttgcacagcg tgctggggac ggcaaatgcc 7620 ccattgagca tctatgaggc catca aaggc gtcgacgggc tcgatgccat ggagccggac 7680 accgcgcccg gcctcccctg ggccctccag gggaaacgcc gtggtgcgtt gattgacttc 7740 gagaacggca cggtcggacc cgaagtcgag gctgccctaa agctcatgga gaaaagagag 7800 taca aatttg cttgccagac cttcctgaaa gacgagattc gtccgatgga aaaagtacgt 7860 gctggcaaga ctcgcattgt cgacgttttg cccgtggaac acattcttta caccaggatg 7920 atgattggca gattctgtgc tcaaatgcac acaaacaatg gaccgcagat tggctcagcg 7980 gtcggttgca atcctgatgt tgattggcaa agatttggca cacattttgc tcagtacaga 8040 aacgtgtggg atgtggacta ttcggccttt gatgctaacc actgcagtga cgcaatgaac 8100 atcatgtttg aggaggtatt tcgcacagac ttcggtttcc acccaaatgc tgagtggatt 8160 ctgaagactc ttgtgaacac ggagcacgcc tatgagaaca aacgtatcac tgttgagggc 8220 gggatgccgt ctggctgttc cgcgaca agc atcatcaaca caattttgaa caacatttat 8280 gtgctctacg ctcttcgtag acactatgag ggagttgagc tggacaccta caccatgatc 8340 tcctacggag atgacatcgt ggttgcaagt gactacgatc tggattttga ggctctcaaa 8400 ccccacttca aatctcttgg tcaaaccatc actccagctg acaaaagcga caaaggtttt 8460 gttcttggtc actccattac cgatgtcact ttcctcaaaa gacacttcca catggactat 8520 ggaactgggt tttacaaacc tgtgatggcc tcaaagaccc tcgaggccat tctctccttt 8580 gcacgccgtg ggaccataca ggagaagttg atctccgtgg caggactcgc cgtccactca 8640 ggacctgacg agtaccggcg tctctttgag cccttcc agg gtctcttcga gattccaagc 8700 tacagatcac tttacctgcg ttgggtgaac gccgtgtgcg gtgacgcata atccctcaga 8760 tgtcacaatt ggcagaaaga ctctgaggcg agcgacgccg taggagtgaa aagcccgaaa 8820 gggcttttcc cgcttcctat tccaaaaaaa aaaaaaaaaa actagttcta gagcggccgc 8880 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaatt gcg cgcttggcgt 8940 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 9000 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 9060 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa c ctgtcgtgc cagctgcatt 9120 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 9180 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 9240 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 9300 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 9360 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 9420 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 9480 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 9540 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 9600 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 9660 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 9720 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 9780 acactagaag gacagtattt gg tatctgcg ctctgctgaa gccagttacc ttcggaaaaa 9840 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 9900 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 9 960 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 10020 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 10080 gtatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 10140 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 10200 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 10260 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 10320 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 10380 gtagt tcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 10440 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 10500 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 10560 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 10620 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 10680 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 10740 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 10800 tctcaaggat cttaccg ctg ttgagatcca gttcgatgta acccactcgt gcacccaact 10860 gatcttcagc atcttttaact ttcaccagcg tttctgggtg agcaaaaca ggaaggcaaa 10920 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 10980 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 11040 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccga aaa gtgccac 11097 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> O1manisa_F primer <400> 8 cttctaaatt ttgacctgct caaat 25 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> O1manisa_R primer <400> 9 ctgtgtgcga gcatccacc 19 <210> 10 <211> 22 <212> DNA < 213> Artificial Sequence <220> <223> PA2_VP1_F primer <400> 10 accacctcca caggtgagtc ag 22 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> PA2_VP1_R primer <400> 11 ctgtttcaca ggtgccacta tc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> JC_VP1_F primer <400> 12 accacttcga caggcgagtc g 21 <210> 13 <211> 21 <212> DNA <213 > Artificial Sequence <220> <223> JC_VP1_R primer<400> 13 ctgctttaca ggtgccacta t 21

Claims (7)

A recombinant foot-and-mouth disease virus having the 646th to 8864th sequence of SEQ ID NO: 6 or the 646th to 8861st sequence of SEQ ID NO: 7. A recombinant plasmid having the sequence of SEQ ID NO: 6 or SEQ ID NO: 7 that produces the recombinant foot-and-mouth disease virus of claim 1. A foot-and-mouth disease O serotype virus antigen obtained by purifying and isolating the recombinant foot-and-mouth disease virus of claim 1. A vaccine composition comprising the recombinant foot-and-mouth disease virus of claim 1 or the foot-and-mouth disease O serotype virus antigen of claim 3. A foot-and-mouth disease diagnostic kit comprising the recombinant foot-and-mouth disease virus of claim 1 or the foot-and-mouth disease virus antigen of claim 3. A method for diagnosing foot-and-mouth disease in non-human ungulates using the foot-and-mouth disease diagnostic kit of claim 6. A method for preventing or treating foot-and-mouth disease in ungulate animals other than humans using a vaccine composition comprising the foot-and-mouth disease recombinant virus of claim 1 or the recombinant foot-and-mouth disease virus antigen of claim 3.