KR102752143B1 - Recombinant foot-and-mouth disease virus type A 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 foot-and-mouth disease recombinant virus that broadly protects against serotype A prevalent in the Asian region or worldwide and a foot-and-mouth disease vaccine composition comprising the same.

Description

{Recombinant foot-and-mouth disease virus type A 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, and more particularly, to a foot-and-mouth disease recombinant virus that broadly protects against serotype A prevalent in Asia or around the world by using a virus produced by artificially inserting and replacing a protective antigen (VP1) region for foot-and-mouth disease type A, and a foot-and-mouth disease vaccine composition comprising the same.

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

The above foot-and-mouth disease pathogen is a single-stranded, positively charged RNA virus belonging to the Piconaviridae family and the Aphthovirus genus, and is classified into seven different serotypes (A, O, C, Asia1, SAT 1, SAT2, SAT3). However, in order to develop all vaccine strains, field strains must be continuously cultured in cells to ensure adequate proliferation, and this method has the disadvantage of being time-consuming.

On the other hand, foot-and-mouth disease virus type A is the virus with the most severe antigenic changes. A22 Iraq is used as the main defensive vaccine strain worldwide, but it may be difficult to use in some regions worldwide due to its low matching rate with the outdoor strain. In the case of type A, it is a virus that is prevalent not only in Asia but also worldwide, and various regional types of viruses with more severe mutations are prevalent, so it is important to use a vaccine strain that can protect broadly against the virus for serotype A, so we tried to develop a vaccine with a broader protective ability.

Patent Registration No. 10-2009270

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

In addition, the present invention aims to provide a method for preventing and treating foot-and-mouth disease using the above foot-and-mouth disease vaccine composition.

To achieve the above purpose, the present invention provides a recombinant virus based on foot-and-mouth disease virus serotype A and a foot-and-mouth disease vaccine composition containing the same as an active ingredient.

In addition, a recombinant plasmid capable of producing the above 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 and 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 is provided using a vaccine composition comprising the recombinant foot-and-mouth disease virus or an antigen isolated and purified from the recombinant foot-and-mouth disease virus.

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 above foot-and-mouth disease diagnostic kit is provided.

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

The recombinant foot-and-mouth disease viruses of the present invention are based on serotype O, and may be those in which VP1 of type O Manisa is replaced with A22iraq70 iso92 VP1 (Genbank. AY593764.1).

Specifically, the recombinant foot-and-mouth disease virus according to the present invention may be foot-and-mouth disease type A viruses, and these may be designated as G1-A22VP1-G7E and G1-A22VP1-G2E, respectively.

More specifically, the above G1-A22VP1-G7E and G1-A22VP1-G2E are viruses purified from sequence numbers 6 and 7, respectively, and their base sequences may have sequence regions from the 646th to the 8864th positions of sequence numbers 6 and from the 646th to the 8861st positions of sequence numbers 7, respectively.

Additionally, the recombinant foot-and-mouth disease virus of the present invention can be administered together with an adjuvant that induces increased immunity.

As used herein, the term "plasmid" means a DNA construct 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 be integrated into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention.

For the purpose of the present invention, it is preferable to use a plasmid vector. A typical plasmid vector that can be used for this purpose has a structure that includes (a) a replication origin that allows efficient replication to include hundreds of plasmid vectors per host cell, (b) a selection marker that allows selection of host cells transformed with the plasmid vector, 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 adaptor or linker according to a conventional method.

The recombinant vector and recombinant foot-and-mouth disease virus of the present invention can be produced by conventional genetic engineering or transformation methods, and an appropriate amount of virus can be obtained by serial passage of the virus formed in a small amount through cell culture.

The cells used in the above cell culture may be derived from at least one type of cell selected from the group consisting of cells of canines, felines, porcines, bovines, deer, giraffes, peccaries, camels, hippopotamuses, equines, tapirs, rhinoceroses, mustels, lagomorphs, rodents and primates, and preferably at least one type selected from the group consisting of goat tongue cells (ZZ-R), hamster kidney cells (BHK-21), black goat kidney cells (BGK), pig kidney cells (IBRS-2) and bovine kidney cells (LFBK).

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

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

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

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

In addition, the vaccine composition can be administered (or injected) to a subject in various forms. Administration can 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 producing the recombinant foot-and-mouth disease virus, the method comprising the following steps:

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

(b) a step of replacing the VP1 to VP4 regions of the Manisa gene of the O serotype of foot-and-mouth disease with the VP1 to VP4 regions of type A;

(c) a step of inserting amino acids from the 140th to the 160th positions of VP1 substituted in step (b);

(d) A step of isolating and purifying the recombinant plasmid obtained through step (c) above.

The sites of VP 1 to 4 substituted in the above step (b) are not limited, but preferably VP2 to VP4 are derived from A/Pocheon among foot-and-mouth disease type A, and VP1 may be substituted with a site derived from A22/Iraq type.

The amino acid substituted in step (c) above may be SGRVRGDLGQLAARVAAQLPA or QNRRGDSGPLVVKPATQLPAS.

The present invention relates to a recombinant foot-and-mouth disease virus and a foot-and-mouth disease vaccine composition comprising the same. Specifically, by using a virus produced by artificially inserting and replacing a protective antigen portion for foot-and-mouth disease type A, a foot-and-mouth disease recombinant virus that broadly protects against serotype A prevalent in the Asian region or worldwide and a foot-and-mouth disease vaccine composition comprising the same can be provided.

Figure 1a shows a genetic schematic diagram of a foot-and-mouth disease recombinant virus according to the present invention.
Figure 1b shows the reactivity according to concentration in the antigen kit of the vaccine of the present invention.
Figure 1c shows the transmission electron microscopy results of the recombinant foot-and-mouth disease virus according to the present invention.
Figures 2a and 2b show the results of evaluating the protective ability against foot-and-mouth disease virus by inoculating mice with two types of foot-and-mouth disease vaccines according to the present invention.
Figure 3 shows the change in antibody levels against representative foot-and-mouth disease type A viruses after immunization of pigs with the vaccine according to the present invention.
Figures 4a to 4f show the results of evaluating the protective ability against three representative types of foot-and-mouth disease virus type A after immunizing pigs with two types of foot-and-mouth disease vaccines according to the present invention.
Figure 5 illustrates a schematic diagram of the backbone of the pOManisa-3B333 vector used for virus production.
Figure 6 is a schematic diagram of the plasmid of A-G1-A22 VP1 (a clone in which A Poc VP4, VP2, VP3 were replaced with A Poc VP4, VP2, VP3 in pO Manisa-3B333 clone and only VP1 was replaced with A22 VP1).
Figure 7 is a schematic diagram of the plasmid producing the A G1-A22VP1-G7E recombinant virus.
Figure 8 is a schematic diagram of the plasmid producing the A G1-A22VP1-G2E recombinant virus.

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

However, the following examples and experimental examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

<Example> Production of recombinant virus according to the present invention

[Experimental Materials and Methods]

Cell and virus preparation

ZZ-R cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM)/F12 (Corning, Union City, NJ, USA) and BHK 21 cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Corning) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Paisley, UK), 100 U/ml penicillin, and 100 mg/ml streptomycin in a 5% CO ₂ atmosphere. BHK 21 suspension cells were maintained in ProVero-1 (Lonza, Belgium) supplemented with dextran sulfate sodium salt and pluronic F-68 and CD-BHK21 supplemented with sodium bicarbonate. The target virus sequences selected for this experiment were o1manisa iso87 (Genbank No. AY593823.1), A/Pocheon/001/KOR/2010 (Genbank No. KC588943.1), A22iraq70 iso92 VP1 (Genbank. AY593764.1), BAN GA Sa-197 2013 (Genbank. KJ754939.1), and A/SKR/4/2018 (Genebank. MK463492.1).

2. Site-directed mutagenesis and subcloning and production of recombinant plasmids

(1) Production of recombinant plasmid pO1Manisa-3B333

The entire genome of foot-and-mouth disease O1Manisa virus (GenBank Accession No. AY593823.1) was amplified by PCR, and the O1Manisa gene was inserted into a plasmid (pBluescript SK II) to construct a recombinant plasmid pO1Manisa-3B333 having the sequence of sequence number 1. For the following experiment, a vector having sequence number 1 was prepared, and the VP2 to VP4 regions of O1manisa iso87 (Genbank No. AY593823.1) were replaced with the VP2 to VP4 regions of the P1 region of A/Pocheon/001/KOR/2010 (Genbank No. KC588943.1) (corresponding to sequence number 2) and the VP1 region of A22iraq70 iso92 VP1 (Genbank. AY593764.1) (corresponding to sequence number 3) to produce a plasmid having sequence number 3.

Afterwards, SGRVRGDLGQLAARVAAQLPA (SEQ ID NO: 9; amino acid sequence corresponding to the base sequence of SEQ ID NO: 5) and QNRRGDSGPLVVKPATQLPAS (SEQ ID NO: 10; amino acid sequence corresponding to the base sequence of SEQ ID NO: 6) were inserted from amino acids 140 to 160 of VP1 of the ASIA regional type genetic subgroups G2 (A/SKR/4/2018 (Genebank. MK463492.1)) and VII (BAN GA Sa-197 2013 (Genbank. KJ754939.1)), respectively, to produce recombinant plasmids.

However, during the production of pO1Manisa-3B333, some genes of O1 Manisa were replaced, as follows. The replaced plasmid was used as a PCR template for amino acid substitution of 3C C142T (5'-ACCATGGATGGAGACACCATG-3 (SEQ ID NO: 11) and 5'-CACTACAATGTCTTTGTAGGTA-3' (SEQ ID NO: 12)). To replace the above 3B1 and 3B2 sites with two 3B3s, a full genome cDNA vector in which both 3B1 and 3B2 were deleted was used, and the synthetic 3B3 dimer gene was amplified with specific primers (5’-AGGACCGACCACAAGCTGAAGGACCTTACGAGGGACCGGT-3’ (SEQ ID NO: 13) and 5’-TCGGTCGGTGGGGCACCACTCTCAGTGACAATCAAGTTCT-3’ (SEQ ID NO: 14)) as a PCR template was inserted into the 3B1 and 3B2 sites.

(2) Production of recombinant plasmid A G1-A22VP1

Among the manufactured recombinant plasmids (pO1Manisa-3B333), the gene encoding the P1 protein, a defensive antigen, was replaced with the gene encoding the P1 protein, a defensive antigen of the Korean regional virus A/Pocheon/001/KOR/2010 (Genbank No. KC588943.1), thereby manufacturing a plasmid (pOm-Apocheon-P1, patent registration 10-1578444, 2015.10.23.) with the recombinant gene inserted. Based on the manufactured pOm-Apocheon-P1, the primers for VP1 amplification of the Middle East vaccine standard strain A22 were the forward primer (5'-ACCACTACCACCGGGGAG-3'; (SEQ ID NO: 15)) and the reverse primer (5'-TTGTTTTGCAGGTGCAATGATC-3'; (SEQ ID NO: 16)), and to amplify the vector, the forward primer (5'-CTTCTAAATTTTGACCTGCTCAAATTGGCGGG-3'; (SEQ ID NO: 17)) and the reverse primer (5'-TTGCGCACGGGGGTCAAT-3'; (SEQ ID NO: 18)) in SEQ ID NO: 4 were used. After amplification, the VP1 gene of the A22 strain and the amplified pOm-Apocheon-P1 for use as a vector were ligated to manufacture the A G1-A22VP1 plasmid of the present invention (SEQ ID NO: 4). Finally, the entire sequence was analyzed to confirm proper cloning (A G1-A22VP1).

(3) Production of recombinant plasmids A G1-A22VP1-G7E and A G1-A22VP1-G2E

In order to insert the 140th to 160th amino acids of VP1 of the manufactured recombinant plasmid (A G1-A22VP1), the A G1-A22VP1 full genome cDNA vector synthesized as a PCR template was used, and the 140th to 160th amino acids of VP1 of VII (BAN GA Sa-197 2013 (Genbank. KJ754939.1) were inserted with specific primers 5'-TCGCGGCGCGAGTCGCTGCTCAACTCCCTGCCGCGAGGGTCGCCGCTCA-3' (SEQ ID NO: 19) and 5'-GCTGTCCCAGGTCACCCCGTACACGCCCACTCGCGAGAGGCCCTAGGTC-3' (SEQ ID NO: 20), thereby constructing A G1-A22VP1-G7E. Finally, through the analysis of the entire base sequence, it was confirmed that it was properly cloned (A G1-A22VP1-G7E). And in order to insert the 140th to 160th amino acids of VP1 in the same manner as the above experimental method, the A G1-A22VP1 full genome cDNA vector synthesized as a PCR template was used and the 140th to 160th amino acids of VP1 of A/SKR/4/2018 (Genebank. MK463492.1) were inserted with specific primers 5'-TGGTGAAACCCGCCACACAGCTCCCTGCCTCCGCGAGGGTCGCCGCTCAG-3' (SEQ ID NO: 21) and 5'-CGAGAGGACCCGAGTCACCTCGCCGGTTTTGCGCGAGAGGCCCTAGGTC-3' (SEQ ID NO: 22) to produce A G1-A22VP1-G2E. The constructed recombinant plasmid was sequenced to confirm the amino acid insertion. Finally, through the analysis of the entire base sequence,

It was confirmed that the plasmids were properly cloned (A G1-A22VP1-G2E). The sequences of the finally manufactured plasmids were confirmed as SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

To isolate and purify the secured recombinant plasmid, CEOS competent cell (Yeastern Biotech, FYE609) was transformed and the plasmid was cloned using LB Broth Base (Invitrogen, 12780052). The cloned plasmid was isolated and purified using Plasmid DNA purification (MN, REF2101/005).

Hereinafter, a recombinant virus (A-G1-A22VP1-G7E) manufactured by inserting SGRVRGDLGQLAARVAAQLPA is referred to as Example 1, and a recombinant virus (A-G1-A22VP1-G2E) manufactured by inserting QNRRGDSGPLVVKPATQLPAS is referred to as Example 2.

3. Virus recovery and cell culture

The recombinant cDNA plasmid was linearized by treatment with the restriction enzyme SpeI (NEB, Beverly, USA) and the infectious cDNA was cultured in BHK-T7-9 cells (a cell line in which T7 RNA polymerase is expressed) at 37°C for 48 h. After that, the virus was harvested by three freeze-thaw cycles and the chimeric virus was used to infect fresh ZZ-R cells. For differentiation from wild-type FMDV and the recombinant virus, structural and nonstructural proteins were identified using the FMDV Rapid Kit (Princeton Bio Meditech Corporation, USA). As a result, the recombinant virus G1-A22VP1-G7E isolated from the plasmid of sequence number 7 was confirmed to have the 646th to 8926th sequence of sequence number 7 (hereinafter, Example 1), and the recombinant virus of A G1-A22VP1-G2E was confirmed to have the 642nd to 8926th sequence of sequence number 8 (hereinafter, Example 2).

4. Virus purification for vaccine antigen

To produce antigens for vaccine manufacturing, the recombinant viruses were cloned using BHK21 or BHK21-suspension cells, which are cells for FMDV production. After 16 h of infection, viruses grown in BHK 21 suspension cells were harvested by freeze-thawing when complete CPE was observed. After virus inactivation with binary ethylene imine (BEI), cell debris was removed. Inactivated viruses from purified cultures were precipitated overnight at 4°C with 7.5% PEG 6000 (81260; Sigma Aldrich, WI, USA) and 2.3% NaCl, and the pellet was resuspended in Tris-KCl (TK) buffer. The resuspended pellet was purified by centrifugation through a 15–45% sucrose gradient in TN buffer at 30,000 rpm for 4 h in an SW41 rotor at 4°C, and the antigen amount was determined by spectrophotometric analysis at 259 nm. The purified antigen was separately blotted onto carbon-coated copper grids and visualized by transmission electron microscopy (Hitachi H7100FA, Tokyo, Japan).

5. Preparation of experimental vaccine

The vaccine was prepared using the method used in a previous study. That is, 15 μg (1 dose) of the 146S antigen purified from Examples 1 and 2, which are the recombinant viruses obtained in the above 3., was mixed with ISA206VG (Seppic, Paris, France). 1:1 (v/v) and 10% aluminum hydroxide gel (Rehyragel® HPA; General Chemical, NJ, USA) and 0.5 μg of saponin were mixed with this mixture to prepare a water-in-oil-in-water vaccine.

6. Virus challenge after vaccination in mature C57BL/6 mice

Seven-week-old C57BL/6 female mice supplied by KOSA-BIO (Republic of Korea) were used in this experiment. Animals were housed in APQA and used with the approval of the Animal Care and Use Committee. The dose groups (dose of vaccination for pigs or cattle (15 μg/dose)) of antigen (1.5, 0.375, 0.093, and 0.023) mixed with ISA 206 (Seppic, France) and 10% aluminum hydroxide gel adjuvant were administered. Seven days post-vaccination (dpv), mice were challenged with 1x10^4 TCID ₅₀ virus 1 day before virus inoculation in the mice to be vaccinated with APOC virus. Mice were challenged with 1x10^4 TCID ₅₀ virus after intraperitoneal administration of 0.125 mg/0.1 mL IFN-aR antibody 1 hour before inoculation with A NEP virus. Mice were challenged with 1x10^5 TCID ₅₀ virus after intraperitoneal administration of 0.125 mg/0.1 mL IFN-aR antibody 1 hour before inoculation with foot-and-mouth disease A-GP virus. Survival was observed for 7 days.

7. ELISA for detection of structural protein antibodies

Structural proteins of FMDV in serum were detected using VDProR® FMDV Type A AB ELISA (MEDIAN Diagnostics, Republic of Korea). For the MEDIAN kit, animals were considered to have an immune response if the sample reflected a value of 0.6 or less.

8. Immunogenicity testing of experimental vaccines in pigs

Pigs (n=5) were vaccinated with 15 μg/ml antigen mixed with ISA 206, saponin, and aluminum hydroxide gel adjuvant. Booster vaccination was given 4 weeks after the primary vaccination, and sera were collected for a total of 12 weeks (0, 7, 14, 21, 28, 42, 68, and 84 dpv).

9. Challenge test of experimental vaccine on pigs

Pigs (n=13) were vaccinated with 15 μg/ml antigen mixed with ISA 206, saponin, and aluminum hydroxide gel adjuvant. After vaccination, sera were collected at 1x10^5 TCID ₅₀ with APOC, A GP, and A NEP viruses (0, 2, 4, 6, and 8 dpc) and saliva was collected daily until day 8. Animals were housed at APQA and used with the approval of its Institutional Animal Care and Use Committee.

10. Virus neutralization test in pigs

After vaccination, serum samples were collected from pigs and heat-inactivated at 56°C for 30 min. The test sera were incubated with 100 TCID ₅₀ of FMDV for 1 h, after which LFBK cells were added to the plates and cultured for 3 days. CPE was determined to determine the titer, which was calculated as the log10 of the reciprocal serum dilution required to neutralize 100 TCID ₅₀ of the virus. FMDV A G1-A22VP1-G7E, A G1-A22VP1-G2E, A POC, A22, A GP, and A NEP were used for VNT in pig sera. Data are expressed as mean ± standard deviation (SD). Differences were considered statistically significant at P < 0.05.

11. Statistical Analysis

Statistical relationships between the vaccinated group and the negative control group were examined. T-tests were performed using GraphPad Prism (ver. 5.0; GraphPad Software, USA) and GraphPad Instant (ver. 3.05; GraphPad Software).

[Experimental Example 1] Characteristics Evaluation of Recombinant FMDV

The whole genome cDNA of O1manisa virus was cloned under the binding site of T7 RNA polymerase. VP4, VP2, VP3 of A/SKR/Pocheon/2010 P1 and VP1 of A22 P1 were replaced for O1manisa, and epitopes (amino acids 140-160) of A/Ban/2013 (GⅦ) and A/SKR/GP/2018 (G2) were added for the production of a new vaccine. The virus was recovered from cells transfected with cDNA (Fig. 1a). 3B1B2 of the viral genome was replaced with 3B3B3, which could be distinguished from the vaccine virus and the FMD wild-type strain using a simple antigen diagnostic kit (lateral flow device; LFD) that allows NSP detection without reaction by the replaced site (Fig. 1b). In addition, the vaccine virus in this experiment confirmed the expected size of FMDV at 25 nm by electron microscopy (Fig. 1c).

[Experimental Example 2] Evaluation of 3-virus vaccine inoculation test in mice after vaccination

After producing a vaccine for Example 1 and immunizing mice (Fig. 2a), a test to determine the protective efficacy against various viruses was conducted. The challenge viruses were A Pocheon, A GP 2018, and A Nep 17 viruses, and the vaccine was diluted to determine the protective efficacy PD _50. The protective titer was confirmed to be >128 mPD ₅₀ for A Pocheon, A GP 2018, and 97 mPD 50 for A Nep 17dp.

After producing a vaccine for Example 2 and immunizing mice (Fig. 2b), a test to determine the protective efficacy against various viruses was conducted. The challenge viruses were A Pocheon, A GP 2018, and A Nep 17 viruses, and the vaccine was diluted to determine the protective efficacy PD _50. The protective titer was confirmed to be >128 mPD ₅₀ for A Pocheon, >128 mPD 50 for A GP 2018, and 32 mPD ₅₀ for A Nep 17dp.

[Experimental Example 3] Comparison of antibodies to various virus strains after immunization in pigs

To compare the titers of antibodies to the three viruses in pigs immunized with the experimental vaccines, a virus neutralization test was performed. The pigs induced a significantly high level of antibody titers (>1:100) three weeks after the first vaccination. After the second vaccination, the immunogenicity tended to increase gradually. The titers of neutralizing antibodies to Sea97/G1, G2, and GⅦ were determined to be high (Fig. 3).

[Experimental Example 4] Test of 3-type virus challenge in pigs after vaccination

After producing a vaccine for Example 1 and immunizing pigs (Figs. 4a to 4c), challenge vaccination against various viruses was performed. The challenge viruses were A Pocheon, A GP 2018, and A Nep 17 viruses. The protective titer was protected against A Pocheon, A GP 2018, and A Nep 17.

After producing a vaccine for Example 2 and immunizing pigs (Figs. 4d to 4f), challenge vaccination against various viruses was performed. The challenge viruses were A Pocheon, A GP 2018, and A Nep 17 viruses. The protective titer was protected against both A Pocheon and A GP 2018. On the other hand, against A Nep 17, one out of four pigs had severe clinical symptoms (6 points), one pig had mild clinical symptoms (1 point), and the remaining two pigs were all protected.

In conclusion, the vaccine strains according to Examples 1 and 2, including the recombinant virus according to the present invention, protected against FMDV type A 3 series viruses in pigs in a target animal experiment, and therefore, it is judged that they can be used as a broad-spectrum vaccine.

<110> Animal and Plant Quarantine Agency <120> Recombinant foot-and-mouth disease virus type A and foot-and-mouth disease vaccine composition comprising the same <130> 21-12108 <160> 22 <170> KoPatentIn 3.0 <210 > 1 <211> 11093 <212> DNA <213> Artificial Sequence <220> <223> pOManisa-3B333 vector <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 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 aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg 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 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 ccgtggggagc 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 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 atgttgatga 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 cacacaacca acactcagaa caacgactgg ttctcgaagc tggccagttc cgctttcagc 2580 ggtcttttcg gcgctcttct cgccgacaag aaaaccgagg agaccactct tctcgaggac 2640 cgcatcctca ctactcgtaa cggacacacc acctcgacaa cccagtcgag cgttggagtc 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 cactgcagtg 2940 ggaaaccagt tcaatggagg atgcctgttg gtggccatgg tgccagaact ttgctccata 3000 cagaagaggg agctgtacca gctcacgctc tttcctcacc agttcatcaa ccctcggacg 3060 aacatgacag cacacatcac tgtgcccttt gttggcgtca accgttatga 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 aacatgttgc cggggcggtt caccaatttt cttgacgtgg ctgaggcgtg ccccacgttt 3420 ctccacttcg agggtgacgt gccatacgtg accacgaaga cggattcaga cagggtgctc 3480 gctcagttcg acttgtcttt ggcagcaaag cacatgtcga acaccttcct tgcaggtctc 3540 gcccagtact acacacagta 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 caaaatgtaca gggatgggtt 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 ttaatgtatt ggacctgatg 4080 caaacccctg ctcacacttt ggtgggagca ctccttcgta ctgccactta ctatttcgct 4140 gacttagagg tggcagtgaa gcacaaggga aacctcacct gggtcccgaa cggggcgcct 4200 gaagcggcgt tggacaacac caccaaccca acagcttacc acaaggcacc 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 gggagatgtg gagtccaacc ctgggccctt cttcttctcc 4620 gacgtcaggt caaatttctc aaaactggta gaaaccatca atcagatgca ggaggacatg 4680 tcaacaaaac acgggcctga ctttaaccgg ttggtgtccg catttgagga attggccact 4740 ggagtgaagg ctatcagggc cggtctcgac gaggccaaac cctggtacaa actcatcaag 4800 ctcctgagcc gcttgtcgtg catggccgct 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 aaagcacgtg acattaacga catattcgcc 5100 attctcaaga acggcgagtg gctggtcaag ctgatccttg ccatccgcga ctggatcaaa 5160 gcgtggatcg cctcagaaga aaagtttgtc accatgacgg acttggtgcc tggtatcctt 5220 gaaaagcagc gggatctcaa cgacccgagt 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 cagcaaagtc atcattgcta ccaccaacct gtactcgggt 5700 ttcaccccga gaacaatggt gtgtcctgac gcgctgaacc ggaggttcca ctttgacatc 5760 gacgtgagtg ccaaggacgg gtacaaagtt aacaaacaaat tggacataat caaagctctt 5820 gaagacaccc acaccaaccc agtggcgatg ttccaatacg actgtgccct tctaaacggt 5880 atggcagttg aaatgaagag aatgcaacag gatatgttca agcctcaacc acccctccag 5940 aacgtgtacc 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 gcggtgccag cactattggt ttcagagaga gaactctccc ggggcacaag 6420 gcgagcgatg acgtgagctc cgagcccgcc aaacccgtgg aggaccgacc acaagctgaa 6480 ggaccttacg agggaccggt gaagaagcct gtcgctttga aagtgaaagc taagaacttg 6540 attgtcactg aggggccata tgaaggacca 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 actgcctacc tcgtacctcg tcacctcttc 6840 gcggagaagt acgacaagat aatgttggac ggtagagcca tgacagacag tgactacaga 6900 gtgtttgagt ttgagattaa agtaaaagga caggacatgc tctcagacgc tgcactcatg 6960 gtgcttcacc gtgggaaccg cgtgagagac atcacgaaac attttcgtga cacagcaaga 7020 atgaagaaag gcacccccgt tgtcggtgtg atcaacaacg ccgacgttgg gagactgatt 7080 ttctctggag aggcccttac ctacaaagac attgtagtga ccatggatgg agacaccatg 7140 ccggggcctgt ttgcctacag agccgccacc aaggctggtt actgcggggg agccgttctc 7200 gccaaggacg gagccgacac attcatcgtt ggcacccact ccgcaggtgg taacggagtt 7260 ggatactgct cgtgcgtgtc caggtccatg ctcctgaaaa tgaaggcaca cattgaccct 7320 gaaccacacc acgaggggtt gattgttgat accagagatg tggaagagcg cgtgcatgtc 7380 atgcgtaaaa ccaagcttgc acccaccgtg gcacacggtg tgtttaaccc tgaatttggt 7440 cccgctgcct tgtccaaacaa ggacccgcgg ctgaacgaag gggttgtcct cgatgaagtc 7500 atcttctcca aacacaaggg agacacgaaa atgtctgagg aggacaaagc gctgttccgc 7560 cgctgcgctg ccgactacgc gtcgcacttg cacagcgtgc tggggacggc aaatgcccca 7620 ttgagcatct atgaggccat caaaggcgtc gacgggctcg atgccatgga gccggacacc 7680 gcgcccggcc tcccctgggc cctccagggg aaacgccgtg gtgcgttgat tgacttcgag 7740 aacggcacgg tcggacccga agtcgaggct gccctaaagc tcatggagaa aagagagtac 7800 aaatttgctt gccagacctt cctgaaagac gagattcgtc cgatggaaaa agtacgtgct 7860 ggcaagactc gcattgtcga cgttttgccc gtggaacaca ttctttacac caggatgatg 7920 attggcagat tctgtgctca aatgcacaca aacaatggac cgcagattgg ctcagcggtc 7980 ggttgcaatc ctgatgttga ttggcaaaga tttggcacac attttgctca gtacagaaac 8040 gtgtgggatg tggactattc ggcctttgat gctaaccact gcagtgacgc aatgaacatc 8100 atgtttgagg aggtatttcg cacagacttc ggtttccacc caaatgctga gtggattctg 8160 aagactcttg tgaacacgga gcacgcctat gagaacaaac gtatcactgt tgagggcggg 8220 atgccgtctg gctgttccgc gacaagcatc atcaacacaa ttttgaacaa 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 actgggtttt acaaacctgt gatggcctca aagaccctcg aggccattct ctcctttgca 8580 cgccgtggga ccatacagga gaagttgatc tccgtggcag gactcgccgt ccactcagga 8640 cctgacgagt accggcgtct ctttgagccc ttccagggtc tcttcgagat tccaagctac 8700 agatcacttt acctgcgttg ggtgaacgcc gtgtgcggtg acgcataatc cctcagatgt 8760 cacaattggc agaaagactc tgaggcgagc gacgccgtag gagtgaaaag cccgaaaggg 8820 cttttcccgc ttcctattcc aaaaaaaaaaa aaaaaaacta gttctagagc ggccgccacc 8880 gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc 8940 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 9000 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 9060 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 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 aaaatcgacg 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 gttaccttcg gaaaaagagt 9840 tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 9900 gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 9960 gtctgacgct cagtggaacg 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 ccgcgagacc cacgctcacc 10260 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 10320 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 10380 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 10440 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 10500 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 10560 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 10620 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 10680 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacggggata ataccgcgcc 10740 acatagcaga actttaaaag tgctcatcat tggaaaaacgt tcttcggggc gaaaactctc 10800 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 10860 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 10920 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 10980 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 11040 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cac 11093 <210> 2 <211> 1572 <212> DNA <213> Artificial Sequence <220> <223> VP4, VP2, VP3 of A Pocheon <400> 2 ggggcagggc aatctagccc tgccaccggg tcacagaacc agtcaggcaa taccggcagc 60 attattaaca actactacat gcagcagtac cagaactcca tggacacaca acttggtgac 120 aacgccatca gcggaggatc caacgagggg tccacggaca caacctctac ccacaaacc 180 aacacccaaa acaatgactg gttctcaaaa ctggcaagtt ccgcgttcac cggtcttttc 240 ggcgcactgc tcgccgacaa gaagaccgaa gagacaactc ttctggagga ccgtatcctc 300 accactcgta atggacacac cacttctaca actcagtcga gtgtgggggt cacctacggg 360 tattcaactg gtgaggacca cgtttctgga cctaacacat caggtttgga gacgcgggtg 420 gtacaagctg aaaggttctt caagaagcac ttgtttgatt ggacgacgga caaacccttt 480 ggtcacgttg aaaagctgga acttcccact gatcacaaag gtgtctacgg acagctggtg 540 gactcctttg catacatgag aaatggctgg gacgtggagg tgtctgctgt tggcaaccag 600 ttcaacggcg ggtgccttct cgtggccatg gtacctgagt ttaaggagtt caccacacgt 660 gaaaagtacc agctcaccct gttcccccac cagttcatta gccccagaac caacatgacc 720 gcgcacatca cggtcccgta ccttggtgtg aacaggtatg accagtacaa caaacacaaa 780 ccctggacgt tggtggtgat ggtggtttca ccacttacca ctagctccat tggtgcatca 840 cagattaagg tctacaccaa tatcgcccct acccacgttc acgtggctgg cgagctcccg 900 tcgaaagagg ggatcgtgcc ggtcgcctgc tcggacgggt acggtggcct ggtgacaaca 960 gaccctaaaa cagctgaccc tgcttacggt atggtgtaca acccacctag gaccaactac 1020 cccgggcggt ttacaaactt gttggacgtg gcagaggcgt gtcccacctt cctctgtttc 1080 gacgacggga aaccgtatgt tgtgacaaga gcggatgagc agcgcctctt ggccaagttt 1140 gacctttccc ttgctgcaaa gcacatgtca aacacctacc tttcagggat agcacagtac 1200 tacgcacagt actctggcac catcaatttg cacttcatgt ttactggttc cactgactca 1260 aaggcccgtt acatggtggc ttacgtcccg cctggcgtga caacgccacc ggacacgcct 1320 gagagagctg cgcactgcat ccacgcagaa tgggacacgg gcctaaactc caaattcact 1380 ttttcaatcc cgtacgtatc tgctgcagat tacgcgtaca cagcgtccga tgtggcagac 1440 acaacaaacg tacagggatg ggtttgcatc taccaaatca cccacgggaa ggccgaacaa 1500 gacactttgg tcgtgtcggt cagcgccggc aaagactttg agctgcgcct ccccattgac 1560 ccccgtgcgc aa 1572 <210> 3 <211> 633 <212> DNA <213> Artificial Sequence <220> <223> VP1 of A22(A22iraq70 iso92) <400> 3 accactacca ccggggagtc tgcagaccct gtcaccacca ctgttgaaaa ctacggcggt 60 gagacacaag tccaacgacg tcagcacacc gacgttactt tcataatgga cagatttgta 120 aagatacaaa acttgaaccc cacacatgtc attgacctca tgcaaaccca ccaacacggg 180 ttggtaggtg ccctgttacg tgctgctacg tactacttct ctgacctgga gattgtggta 240 cgccatgacg gtaacctaac ctgggtaccc aatggagcac ccgaggcagc tctgtctaac 300 acgggcaacc ccaccgccta cctcaaggca ccatttacga ggctcgcgct cccctacacc 360 gcgccacacacc gcgtgttggc aacagtgtac aacgggacga gcaagtactc cgcaggtggt 420 acgggcagac ggggcgacct agggcctctc gcggcgaggg tcgccgctca gcttcctgct 480 tctttcaact ttggtgcaat tcaagccacg accatccacg agctcctcgt gcgcatgaag 540 cgtgccgaac tctactgccc cagaccactg ttggcagtgg aggtgtcgtc tcaagacaga 600 cacaaacaga agatcattgc acctgcaaaa caa 633 <210> 4 <211> 11099 <212> DNA <213> Artificial Sequence <220> <223> A-G1-A22 VP1 plasmid <400> 4 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 gggggcaggg caatctagcc ctgccaccgg gtcacagaac cagtcaggca 2400 ataccggcag cattattaac aactactaca tgcagcagta ccagaactcc atggacacac 2460 aacttggtga caacgccatc agcggaggat ccaacgaggg gtccacggac acaacctcta 2520 cccacacaac caacacccaa aacaatgact ggttctcaaa actggcaagt tccgcgttca 2580 ccggtctttt cggcgcactg ctcgccgaca agaagaccga agagacaact cttctggagg 2640 accgtatcct caccactcgt aatggacaca ccacttctac aactcagtcg agtgtggggg 2700 tcacctacgg gtattcaact ggtgaggacc acgtttctgg acctaacaca tcaggtttgg 2760 agacgcgggt ggtacaagct gaaaggttct tcaagaagca cttgtttgat tggacgacgg 2820 acaaaccctt tggtcacgtt gaaaagctgg aacttcccac tgatcacaaa ggtgtctacg 2880 gacagctggt ggactccttt gcatacatga gaaatggctg ggacgtggag gtgtctgctg 2940 ttggcaacca gttcaacggc gggtgccttc tcgtggccat ggtacctgag tttaaggagt 3000 tcaccacacg tgaaaagtac cagctcaccc tgttccccca ccagttcatt agccccagaa 3060 ccaacatgac cgcgcacatc acggtcccgt accttggtgt gaacaggtat gaccagtaca 3120 acaaacacaa accctggacg ttggtggtga tggtggtttc accacttacc actagctcca 3180 ttggtgcatc acagattaag gtctacacca atatcgcccc tacccacgtt cacgtggctg 3240 gcgagctccc gtcgaaagag gggatcgtgc cggtcgcctg ctcggacggg tacggtggcc 3300 tggtgacaac agaccctaaa acagctgacc ctgcttacgg tatggtgtac aacccaccta 3360 ggaccaacta ccccgggcgg tttacaaact tgttggacgt ggcagaggcg tgtcccacct 3420 tcctctgttt cgacgacggg aaaccgtatg ttgtgacaag agcggatgag cagcgcctct 3480 tggccaagtt tgacctttcc cttgctgcaa agcacatgtc aaacacctac ctttcaggga 3540 tagcacagta ctacgcacag tactctggca ccatcaattt gcacttcatg tttactggtt 3600 ccactgactc aaaggcccgt tacatggtgg cttacgtccc gcctggcgtg acaacgccac 3660 cggacacgcc tgagagagct gcgcactgca tccacgcaga atgggacacg ggcctaaact 3720 ccaaattcac tttttcaatc ccgtacgtat ctgctgcaga ttacgcgtac acagcgtccg 3780 atgtggcaga cacaacaaac gtacagggat gggtttgcat ctaccaaatc acccacggga 3840 aggccgaaca agacactttg gtcgtgtcgg tcagcgccgg caaagacttt gagctgcgcc 3900 tccccattga cccccgtgcg caaaccacta ccaccgggga gtctgcagac cctgtcacca 3960 ccactgttga aaactacggc ggtgagacac aagtccaacg acgtcagcac accgacgtta 4020 ctttcataat ggacagattt gtaaagatac aaaacttgaa ccccacacat gtcattgacc 4080 tcatgcaaac ccaccaacac gggttggtag gtgccctgtt acgtgctgct acgtactact 4140 tctctgacct ggagatgtg gtacgccatg acggtaacct aacctgggta cccaatggag 4200 cacccgaggc agctctgtct aacacgggca accccaccgc ctacctcaag gcaccattta 4260 cgaggctcgc gctcccctac accgcgccac accgcgtgtt ggcaacagtg tacaacggga 4320 cgagcaagta ctccgcaggt ggtacgggca gacggggcga cctagggcct ctcgcggcga 4380 gggtcgccgc tcagcttcct gcttctttca actttggtgc aattcaagcc acgaccatcc 4440 acgagctcct cgtgcgcatg aagcgtgccg aactctactg ccccagacca ctgttggcag 4500 tggaggtgtc gtctcaagac agacacaaac agaagatcat tgcacctgca aaacaacttc 4560 taaattttga cctgctcaaa ttggcgggag atgtggagtc caaccctggg cccttcttct 4620 tctccgacgt caggtcaaat ttctcaaaac tggtagaaac catcaatcag atgcaggagg 4680 acatgtcaac aaaacacggg cctgacttta accggttggt gtccgcattt gaggaattgg 4740 ccactggagt gaaggctatc agggccggtc tcgacgaggc caaaccctgg tacaaactca 4800 tcaagctcct gagccgcttg tcgtgcatgg ccgctgtagc agcacggtca aaggacccag 4860 tccttgtggc catcatgctg gctgacaccg gtcttgagat tctggacagc acctttgtcg 4920 tgaagaagat ctccgactcg ctctccagtc tctttcacgt gccggccccc gtcttcagtt 4980 tcggagcccc gattctgttg gccgggttgg tcaaagtcgc ctcgagtttc ttccggtcca 5040 cacccgaaga ccttgagaga gcagaaaaac agctcaaagc acgtgacatt aacgacatat 5100 tcgccattct caagaacggc gagtggctgg tcaagctgat ccttgccatc cgcgactgga 5160 tcaaagcgtg gatcgcctca gaagaaaagt ttgtcaccat gacggacttg gtgcctggta 5220 tccttgaaaa gcagcgggat ctcaacgacc cgagtaagta caaggaagcc aaggagtggc 5280 tcgacaacgc gcgccaggcg tgtttgaaga gcgggaacgt tcacattgcc aatttgtgca 5340 aagtggtcgc cccggcaccc agcaagtcga gacccgaacc cgtggtcgtt tgcctccgcg 5400 gcaaatccgg ccagggggaag agtttccttg cgaacgtgct cgcgcaagca atctccaccc 5460 acttcaccgg cagaactgat tcggtttggt actgcccgcc tgaccctgac cacttcgacg 5520 gttacaacca gcagaccgtt gtcgtgatgg acgatttggg ccagaacccc gatggcaagg 5580 acttcaagta cttcgcccag atggtttcga ccacggggtt catcccgccc atggcctcgc 5640 ttgaggacaa aggcaagcct ttcaacagca aagtcatcat tgctaccacc aacctgtact 5700 cgggtttcac cccgagaaca atggtgtgtc ctgacgcgct gaaccggagg ttccactttg 5760 acatcgacgt gagtgccaag gacgggtaca aagttaacaa caaattggac ataatcaaag 5820 ctcttgaaga cacccacacc aacccagtgg cgatgttcca atacgactgt gcccttctaa 5880 acggtatggc agttgaaatg aagagaatgc aacaggatat gttcaagcct caaccacccc 5940 tccagaacgt gtaccaactc gttcacgagg tgattgaacg ggtcgagctc cacgagaagg 6000 tgtcgagcca cccgattttc aaacagatat caattccttc ccaaaagtct gtgttgtact 6060 tcctcattga gaaaggccaa cacgaagcag caattgaatt ctttgaggga atggtgcatg 6120 actccatcaa ggaagagctc cggcccctca tccaacagac ctcatttgtg aaacgcgctt 6180 ttaagcgcct gaaggaaaac tttgagactg ttgccctgtg tttgactctt ttggcaaaca 6240 tagtgatcat gatccgcgag actcgcaaga gacaacagat ggtggacgat gcagtgaatg 6300 actacattga gaaggcaaac atcaccacag atgacaagac tcttgacgag gcggaaaaga 6360 accctctaga gaccagcggt gccagcacta ttggtttcag agagagaact ctcccggggc 6420 acaaggcgag cgatgacgtg agctccgagc ccgccaaacc cgtggaggac cgaccacaag 6480 ctgaaggacc ttacgaggga ccggtgaaga agcctgtcgc tttgaaagtg aaagctaaga 6540 acttgattgt cactgagggg ccatatgaag gaccagtgaa gaaacctgtc gctttgaaag 6600 tgaaagcaaa agccccgatt gtcactgaag gaccctacga gggaccggtg aagaagcctg 6660 tcgctttgaa agtgaaagcc aagaacttga ttgtcactga gagtggtgcc ccaccgaccg 6720 acttgcagaa gatggtcatg ggcaacacta agcctgttga gctcatcctc gacgggaaga 6780 cggtagccat ctgctgtgct accggagtgt ttggcactgc ctacctcgta cctcgtcacc 6840 tcttcgcgga gaagtacgac aagataatgt tggacggtag agccatgaca gacagtgact 6900 acagagtgtt tgagtttgag attaaagtaa aaggacagga catgctctca gacgctgcac 6960 tcatggtgct tcaccgtggg aaccgcgtga gagacatcac gaaacatttt cgtgacacag 7020 caagaatgaa gaaaggcacc cccgttgtcg gtgtgatcaa caacgccgac gttgggagac 7080 tgattttctc tggagaggcc cttacctaca aagacattgt agtgaccatg gatggagaca 7140 ccatgccggg cctgtttgcc tacagagccg ccaccaaggc tggttactgc gggggagccg 7200 ttctcgccaa ggacggagcc gacacattca tcgttggcac ccactccgca ggtggtaacg 7260 gagttggata ctgctcgtgc gtgtccaggt ccatgctcct gaaaatgaag gcacacattg 7320 accctgaacc acaccacgag gggttgattg ttgataccag agatgtggaa gagcgcgtgc 7380 atgtcatgcg taaaaccaag cttgcaccca ccgtggcaca cggtgtgttt aaccctgaat 7440 ttggtcccgc tgccttgtcc aacaaggacc cgcggctgaa cgaaggggtt gtcctcgatg 7500 aagtcatctt ctccaaacac aagggagaca cgaaaatgtc tgaggaggac aaagcgctgt 7560 tccgccgctg cgctgccgac tacgcgtcgc acttgcacag cgtgctgggg acggcaaatg 7620 ccccattgag catctatgag gccatcaaag gcgtcgacgg gctcgatgcc atggagccgg 7680 acaccgcgcc cggcctcccc tgggccctcc aggggaaacg ccgtggtgcg ttgattgact 7740 tcgagaacgg cacggtcgga cccgaagtcg aggctgccct aaagctcatg gagaaaagag 7800 agtacaaatt tgcttgccag accttcctga aagacgagat tcgtccgatg gaaaaaagtac 7860 gtgctggcaa gactcgcatt gtcgacgttt tgcccgtgga acacattctt tacaccagga 7920 tgatgattgg cagattctgt gctcaaatgc acacaaaacaa tggaccgcag attggctcag 7980 cggtcggttg caatcctgat gttgattggc aaagatttgg cacacatttt gctcagtaca 8040 gaaacgtgtg ggatgtggac tattcggcct ttgatgctaa ccactgcagt gacgcaatga 8100 acatcatgtt tgaggaggta tttcgcacag acttcggttt ccacccaaat gctgagtgga 8160 ttctgaagac tcttgtgaac acggagcacg cctatgagaa caaacgtatc actgttgagg 8220 gcgggatgcc gtctggctgt tccgcgacaa gcatcatcaa cacaattttg aacaacatt 8280 atgtgctcta cgctcttcgt agacactatg agggagttga gctggacacc tacaccatga 8340 tctcctacgg agatgacatc gtggttgcaa gtgactacga tctggatttt gaggctctca 8400 aaccccactt caaatctctt ggtcaaacca tcactccagc tgacaaaagc gacaaaggtt 8460 ttgttcttgg tcactccatt accgatgtca ctttcctcaa aagacacttc cacatggact 8520 atggaactgg gttttacaaa cctgtgatgg cctcaaagac cctcgaggcc attctctcct 8580 ttgcacgccg tgggaccata caggagaagt tgatctccgt ggcaggactc gccgtccact 8640 caggacctga cgagtaccgg cgtctctttg agcccttcca gggtctcttc gagattccaa 8700 gctacagatc actttacctg cgttgggtga acgccgtgtg cggtgacgca taatccctca 8760 gatgtcacaa ttggcagaaa gactctgagg cgagcgacgc cgtaggagtg aaaagcccga 8820 aagggctttt cccgcttcct attccaaaaa aaaaaaaaaaa aaactagttc tagagcggcc 8880 gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc 8940 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 9000 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 9060 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 9120 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 9180 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 9240 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 9300 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 9360 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 9420 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 9480 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 9540 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 9600 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 9660 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 9720 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 9780 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 9840 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 9900 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 9960 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 10020 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 10080 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 10140 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 10200 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 10260 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 10320 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 10380 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 10440 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 10500 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 10560 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 10620 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 10680 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 10740 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 10800 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 10860 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 10920 aaaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 10980 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 11040 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac 11099 <210> 5 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> G7E epitope <400> 5 agtgggcgtg tacggggtga cctgggacag ctcgcggcgc gagtcgctgc tcaactccct 60 gcc 63 <210> 6 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> G2E epitope <400> 6 caaaaccggc gaggtgactc gggtcctctc gtggtgaaac ccgccacaca gctccctgcc 60 tcc 63 <210> 7 <211> 11162 <212> DNA <213> Artificial Sequence <220> <223> A G1-A22VP1-G7E plasmid <400> 7 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 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 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 gggggcaggg caatctagcc ctgccaccgg gtcacagaac cagtcaggca 2400 ataccggcag cattattaac aactactaca tgcagcagta ccagaactcc atggacacac 2460 aacttggtga caacgccatc agcggaggat ccaacgaggg gtccacggac acaacctcta 2520 cccacacaac caacacccaa aacaatgact ggttctcaaa actggcaagt tccgcgttca 2580 ccggtctttt cggcgcactg ctcgccgaca agaagaccga agagacaact cttctggagg 2640 accgtatcct caccactcgt aatggacaca ccacttctac aactcagtcg agtgtggggg 2700 tcacctacgg gtattcaact ggtgaggacc acgtttctgg acctaacaca tcaggtttgg 2760 agacgcgggt ggtacaagct gaaaggttct tcaagaagca cttgtttgat tggacgacgg 2820 acaaaccctt tggtcacgtt gaaaagctgg aacttcccac tgatcacaaa ggtgtctacg 2880 gacagctggt ggactccttt gcatacatga gaaatggctg ggacgtggag gtgtctgctg 2940 ttggcaacca gttcaacggc gggtgccttc tcgtggccat ggtacctgag tttaaggagt 3000 tcaccacacg tgaaaagtac cagctcaccc tgttccccca ccagttcatt agccccagaa 3060 ccaacatgac cgcgcacatc acggtcccgt accttggtgt gaacaggtat gaccagtaca 3120 acaaacacaa accctggacg ttggtggtga tggtggtttc accacttacc actagctcca 3180 ttggtgcatc acagattaag gtctacacca atatcgcccc tacccacgtt cacgtggctg 3240 gcgagctccc gtcgaaagag gggatcgtgc cggtcgcctg ctcggacggg tacggtggcc 3300 tggtgacaac agaccctaaa acagctgacc ctgcttacgg tatggtgtac aacccaccta 3360 ggaccaacta ccccgggcgg tttacaaact tgttggacgt ggcagaggcg tgtcccacct 3420 tcctctgttt cgacgacggg aaaccgtatg ttgtgacaag agcggatgag cagcgcctct 3480 tggccaagtt tgacctttcc cttgctgcaa agcacatgtc aaacacctac ctttcaggga 3540 tagcacagta ctacgcacag tactctggca ccatcaattt gcacttcatg tttactggtt 3600 ccactgactc aaaggcccgt tacatggtgg cttacgtccc gcctggcgtg acaacgccac 3660 cggacacgcc tgagagagct gcgcactgca tccacgcaga atgggacacg ggcctaaact 3720 ccaaattcac tttttcaatc ccgtacgtat ctgctgcaga ttacgcgtac acagcgtccg 3780 atgtggcaga cacaacaaac gtacagggat gggtttgcat ctaccaaatc acccacggga 3840 aggccgaaca agacactttg gtcgtgtcgg tcagcgccgg caaagacttt gagctgcgcc 3900 tccccattga cccccgtgcg caaaccacta ccaccgggga gtctgcagac cctgtcacca 3960 ccactgttga aaactacggc ggtgagacac aagtccaacg acgtcagcac accgacgtta 4020 ctttcataat ggacagattt gtaaagatac aaaacttgaa ccccacacat gtcattgacc 4080 tcatgcaaac ccaccaacac gggttggtag gtgccctgtt acgtgctgct acgtactact 4140 tctctgacct ggagatgtg gtacgccatg acggtaacct aacctgggta cccaatggag 4200 cacccgaggc agctctgtct aacacgggca accccaccgc ctacctcaag gcaccattta 4260 cgaggctcgc gctcccctac accgcgccac accgcgtgtt ggcaacagtg tacaacggga 4320 cgagcaagta ctccgcaggt ggtacgggca gacggggcga cctagggcct ctcgcgagtg 4380 ggcgtgtacg gggtgacctg ggacagctcg cggcgcgagt cgctgctcaa ctccctgccg 4440 cgagggtcgc cgctcagctt cctgcttctt tcaactttgg tgcaattcaa gccacgacca 4500 tccacgagct cctcgtgcgc atgaagcgtg ccgaactcta ctgccccaga ccactgttgg 4560 cagtggaggt gtcgtctcaa gacagacaca aacagaagat cattgcacct gcaaaacaac 4620 ttctaaattt tgacctgctc aaattggcgg gagatgtgga gtccaaccct gggcccttct 4680 tcttctccga cgtcaggtca aatttctcaa aactggtaga aaccatcaat cagatgcagg 4740 aggacatgtc aacaaaacac gggcctgact ttaaccggtt ggtgtccgca tttgaggaat 4800 tggccactgg agtgaaggct atcagggccg gtctcgacga ggccaaaccc tggtacaaac 4860 tcatcaagct cctgagccgc ttgtcgtgca tggccgctgt agcagcacgg tcaaaggacc 4920 cagtccttgt ggccatcatg ctggctgaca ccggtcttga gattctggac agcacctttg 4980 tcgtgaagaa gatctccgac tcgctctcca gtctctttca cgtgccggcc cccgtcttca 5040 gtttcggagc cccgattctg ttggccgggt tggtcaaagt cgcctcgagt ttcttccggt 5100 ccacacccga agaccttgag agagcagaaa aacagctcaa agcacgtgac attaacgaca 5160 tattcgccat tctcaagaac ggcgagtggc tggtcaagct gatccttgcc atccgcgact 5220 ggatcaaagc gtggatcgcc tcagaagaaa agtttgtcac catgacggac ttggtgcctg 5280 gtatccttga aaagcagcgg gatctcaacg acccgagtaa gtacaaggaa gccaaggagt 5340 ggctcgacaa cgcgcgccag gcgtgtttga agagcgggaa cgttcacatt gccaatttgt 5400 gcaaagtggt cgccccggca cccagcaagt cgagacccga acccgtggtc gtttgcctcc 5460 gcggcaaatc cggccagggg aagagtttcc ttgcgaacgt gctcgcgcaa gcaatctcca 5520 cccacttcac cggcagaact gattcggttt ggtactgccc gcctgaccct gaccacttcg 5580 acggttacaa ccagcagacc gttgtcgtga tggacgattt gggccagaac cccgatggca 5640 aggacttcaa gtacttcgcc cagatggttt cgaccacggg gttcatcccg cccatggcct 5700 cgcttgagga caaaggcaag cctttcaaca gcaaagtcat cattgctacc accaacctgt 5760 actcgggttt caccccgaga acaatggtgt gtcctgacgc gctgaaccgg aggttccact 5820 ttgacatcga cgtgagtgcc aaggacgggt acaaagttaa caacaaattg gacataatca 5880 aagctcttga agacacccac accaacccag tggcgatgtt ccaatacgac tgtgcccttc 5940 ggcagttgaa atgaagagaa tgcaacagga tatgttcaag cctcaaccac 6000 ccctccagaa cgtgtaccaa ctcgttcacg aggtgattga acgggtcgag ctccacgaga 6060 aggtgtcgag ccacccgatt ttcaaacaga tatcaattcc ttcccaaaag tctgtgttgt 6120 acttcctcat tgagaaaggc caacacgaag cagcaattga attctttgag ggaatggtgc 6180 atgactccat caaggaagag ctccggcccc tcatccaaca gacctcattt gtgaaacgcg 6240 cttttaagcg cctgaaggaa aactttgaga ctgttgccct gtgtttgact cttttggcaa 6300 acatagtgat catgatccgc gagactcgca agagacaaca gatggtggac gatgcagtga 6360 atgactacat tgagaaggca aacatcacca cagatgacaa gactcttgac gaggcggaaa 6420 agaaccctct agagaccagc ggtgccagca ctattggttt cagagagaga actctcccgg 6480 ggcacaaggc gagcgatgac gtgagctccg agcccgccaa acccgtggag gaccgaccac 6540 aagctgaagg accttacgag ggaccggtga agaagcctgt cgctttgaaa gtgaaagcta 6600 agaacttgat tgtcactgag gggccatatg aaggaccagt gaagaaacct gtcgctttga 6660 aagtgaaagc aaaagccccg attgtcactg aaggacccta cgagggaccg gtgaagaagc 6720 ctgtcgcttt gaaagtgaaa gccaagaact tgattgtcac tgagagtggt gccccaccga 6780 ccgacttgca gaagatggtc atgggcaaca ctaagcctgt tgagctcatc ctcgacggga 6840 agacggtagc catctgctgt gctaccggag tgtttggcac tgcctacctc gtacctcgtc 6900 acctcttcgc ggagaagtac gacaagataa tgttggacgg tagagccatg acagacagtg 6960 actacagagt gtttgagttt gagattaaag taaaaggaca ggacatgctc tcagacgctg 7020 cactcatggt gcttcaccgt gggaaccgcg tgagagacat cacgaaacat tttcgtgaca 7080 cagcaagaat gaagaaaggc acccccgttg tcggtgtgat caacaacgcc gacgttggga 7140 gactgatttt ctctggagag gcccttacct acaaagacat tgtagtgacc atggatggag 7200 acaccatgcc gggcctgttt gcctacagag ccgccaccaa ggctggttac tgcgggggag 7260 ccgttctcgc caaggacgga gccgacacat tcatcgttgg cacccactcc gcaggtggta 7320 acggagttgg atactgctcg tgcgtgtcca ggtccatgct cctgaaaatg aaggcacaca 7380 ttgaccctga accacaccac gaggggttga ttgttgatac cagagatgtg gaagagcgcg 7440 tgcatgtcat gcgtaaaacc aagcttgcac ccaccgtggc acacggtgtg tttaaccctg 7500 aatttggtcc cgctgccttg tccaaacaagg acccgcggct gaacgaaggg gttgtcctcg 7560 atgaagtcat cttctccaaa cacaagggag acacgaaaat gtctgaggag gacaaagcgc 7620 tgttccgccg ctgcgctgcc gactacgcgt cgcacttgca cagcgtgctg gggacggcaa 7680 atgccccatt gagcatctat gaggccatca aaggcgtcga cgggctcgat gccatggagc 7740 cggacaccgc gcccggcctc ccctgggccc tccaggggaa acgccgtggt gcgttgattg 7800 acttcgagaa cggcacggtc ggacccgaag tcgaggctgc cctaaagctc atggagaaaa 7860 gagagtacaa atttgcttgc cagaccttcc tgaaagacga gattcgtccg atggaaaaag 7920 tacgtgctgg caagactcgc attgtcgacg ttttgcccgt ggaacacatt ctttacacca 7980 ggatgatgat tggcagattc tgtgctcaaa tgcacacaaa caatggaccg cagattggct 8040 cagcggtcgg ttgcaatcct gatgttgatt ggcaaagatt tggcacacat tttgctcagt 8100 acagaaacgt gtgggatgtg gactattcgg cctttgatgc taaccactgc agtgacgcaa 8160 tgaacatcat gtttgaggag gtatttcgca cagacttcgg tttccaccca aatgctgagt 8220 ggattctgaa gactcttgtg aacacggagc acgcctatga gaacaaacgt atcactgttg 8280 agggcgggat gccgtctggc tgttccgcga caagcatcat caacacaatt ttgaacaaca 8340 tttatgtgct ctacgctctt cgtagacact atgagggagt tgagctggac acctacacca 8400 tgatctccta cggagatgac atcgtggttg caagtgacta cgatctggat tttgaggctc 8460 tcaaacccca cttcaaatct cttggtcaaa ccatcactcc agctgacaaa agcgacaaag 8520 gttttgttct tggtcactcc attaccgatg tcactttcct caaaagacac ttccacatgg 8580 actatggaac tgggttttac aaacctgtga tggcctcaaa gaccctcgag gccattctct 8640 cctttgcacg ccgtgggacc atacaggaga agttgatctc cgtggcagga ctcgccgtcc 8700 actcaggacc tgacgagtac cggcgtctct ttgagccctt ccagggtctc ttcgagattc 8760 caagctacag atcactttac ctgcgttggg tgaacgccgt gtgcggtgac gcataatccc 8820 tcagatgtca caattggcag aaagactctg aggcgagcga cgccgtagga gtgaaaagcc 8880 cgaaagggct tttcccgctt cctattccaa aaaaaaaaaaa aaaaaactag ttctagagcg 8940 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt 9000 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 9060 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 9120 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 9180 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 9240 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 9300 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 9360 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 9420 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 9480 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 9540 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 9600 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 9660 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 9720 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 9780 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 9840 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 9900 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 9960 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 10020 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 10080 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 10140 ctaaagtata tatgagtaaa cttggtctga cagttaaccaa tgcttaatca gtgaggcacc 10200 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 10260 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 10320 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 10380 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 10440 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 10500 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 10560 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 10620 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 10680 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 10740 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacggggataa 10800 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 10860 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 10920 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 10980 gcaaaatgcc gcaaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 11040 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 11100 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 11160 ac 11162 <210> 8 <211> 11162 <212> DNA <213> Artificial Sequence <220> <223> A G1-A22VP1-G2E plasmid <400> 8 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 gggggcaggg caatctagcc ctgccaccgg gtcacagaac cagtcaggca 2400 ataccggcag cattattaac aactactaca tgcagcagta ccagaactcc atggacacac 2460 aacttggtga caacgccatc agcggagggat ccaacgaggg gtccacggac acaacctcta 2520 cccacacaac caacacccaa aacaatgact ggttctcaaa actggcaagt tccgcgttca 2580 ccggtctttt cggcgcactg ctcgccgaca agaagaccga agagacaact cttctggagg 2640 accgtatcct caccactcgt aatggacaca ccacttctac aactcagtcg agtgtggggg 2700 tcacctacgg gtattcaact ggtgaggacc acgtttctgg acctaacaca tcaggtttgg 2760 agacgcgggt ggtacaagct gaaaggttct tcaagaagca cttgtttgat tggacgacgg 2820 acaaaccctt tggtcacgtt gaaaagctgg aacttcccac tgatcacaaa ggtgtctacg 2880 gacagctggt ggactccttt gcatacatga gaaatggctg ggacgtggag gtgtctgctg 2940 ttggcaacca gttcaacggc gggtgccttc tcgtggccat ggtacctgag tttaaggagt 3000 tcaccacacg tgaaaagtac cagctcaccc tgttccccca ccagttcatt agccccagaa 3060 ccaacatgac cgcgcacatc acggtcccgt accttggtgt gaacaggtat gaccagtaca 3120 acaaacacaa accctggacg ttggtggtga tggtggtttc accacttacc actagctcca 3180 ttggtgcatc acagattaag gtctacacca atatcgcccc tacccacgtt cacgtggctg 3240 gcgagctccc gtcgaaagag gggatcgtgc cggtcgcctg ctcggacggg tacggtggcc 3300 tggtgacaac agaccctaaa acagctgacc ctgcttacgg tatggtgtac aacccaccta 3360 ggaccaacta ccccgggcgg tttacaaact tgttggacgt ggcagaggcg tgtcccacct 3420 tcctctgttt cgacgacggg aaaccgtatg ttgtgacaag agcggatgag cagcgcctct 3480 tggccaagtt tgacctttcc cttgctgcaa agcacatgtc aaacacctac ctttcaggga 3540 tagcacagta ctacgcacag tactctggca ccatcaattt gcacttcatg tttactggtt 3600 ccactgactc aaaggcccgt tacatggtgg cttacgtccc gcctggcgtg acaacgccac 3660 cggacacgcc tgagagagct gcgcactgca tccacgcaga atgggacacg ggcctaaact 3720 ccaaattcac tttttcaatc ccgtacgtat ctgctgcaga ttacgcgtac acagcgtccg 3780 atgtggcaga cacaacaaac gtacagggat gggtttgcat ctaccaaatc acccacggga 3840 aggccgaaca agacactttg gtcgtgtcgg tcagcgccgg caaagacttt gagctgcgcc 3900 tccccattga cccccgtgcg caaaccacta ccaccgggga gtctgcagac cctgtcacca 3960 ccactgttga aaactacggc ggtgagacac aagtccaacg acgtcagcac accgacgtta 4020 ctttcataat ggacagattt gtaaagatac aaaacttgaa ccccacacat gtcattgacc 4080 tcatgcaaac ccaccaacac gggttggtag gtgccctgtt acgtgctgct acgtactact 4140 tctctgacct ggagatgtg gtacgccatg acggtaacct aacctgggta cccaatggag 4200 cacccgaggc agctctgtct aacacgggca accccaccgc ctacctcaag gcaccattta 4260 cgaggctcgc gctcccctac accgcgccac accgcgtgtt ggcaacagtg tacaacggga 4320 cgagcaagta ctccgcaggt ggtacgggca gacggggcga cctagggcct ctcgcgcaaa 4380 accggcgagg tgactcgggt cctctcgtgg tgaaacccgc cacacagctc cctgcctccg 4440 cgagggtcgc cgctcagctt cctgcttctt tcaactttgg tgcaattcaa gccacgacca 4500 tccacgagct cctcgtgcgc atgaagcgtg ccgaactcta ctgccccaga ccactgttgg 4560 cagtggaggt gtcgtctcaa gacagacaca aacagaagat cattgcacct gcaaaacaac 4620 ttctaaattt tgacctgctc aaattggcgg gagatgtgga gtccaaccct gggcccttct 4680 tcttctccga cgtcaggtca aatttctcaa aactggtaga aaccatcaat cagatgcagg 4740 aggacatgtc aacaaaacac gggcctgact ttaaccggtt ggtgtccgca tttgaggaat 4800 tggccactgg agtgaaggct atcagggccg gtctcgacga ggccaaaccc tggtacaaac 4860 tcatcaagct cctgagccgc ttgtcgtgca tggccgctgt agcagcacgg tcaaaggacc 4920 cagtccttgt ggccatcatg ctggctgaca ccggtcttga gattctggac agcacctttg 4980 tcgtgaagaa gatctccgac tcgctctcca gtctctttca cgtgccggcc cccgtcttca 5040 gtttcggagc cccgattctg ttggccgggt tggtcaaagt cgcctcgagt ttcttccggt 5100 ccacacccga agaccttgag agagcagaaa aacagctcaa agcacgtgac attaacgaca 5160 tattcgccat tctcaagaac ggcgagtggc tggtcaagct gatccttgcc atccgcgact 5220 ggatcaaagc gtggatcgcc tcagaagaaa agtttgtcac catgacggac ttggtgcctg 5280 gtatccttga aaagcagcgg gatctcaacg acccgagtaa gtacaaggaa gccaaggagt 5340 ggctcgacaa cgcgcgccag gcgtgtttga agagcgggaa cgttcacatt gccaatttgt 5400 gcaaagtggt cgccccggca cccagcaagt cgagacccga acccgtggtc gtttgcctcc 5460 gcggcaaatc cggccagggg aagagtttcc ttgcgaacgt gctcgcgcaa gcaatctcca 5520 cccacttcac cggcagaact gattcggttt ggtactgccc gcctgaccct gaccacttcg 5580 acggttacaa ccagcagacc gttgtcgtga tggacgattt gggccagaac cccgatggca 5640 aggacttcaa gtacttcgcc cagatggttt cgaccacggg gttcatcccg cccatggcct 5700 cgcttgagga caaaggcaag cctttcaaca gcaaagtcat cattgctacc accaacctgt 5760 actcgggttt caccccgaga acaatggtgt gtcctgacgc gctgaaccgg aggttccact 5820 ttgacatcga cgtgagtgcc aaggacgggt acaaagttaa caacaaattg gacataatca 5880 aagctcttga agacacccac accaacccag tggcgatgtt ccaatacgac tgtgcccttc 5940 taaacggtat ggcagttgaa atgaagagaa tgcaacagga tatgttcaag cctcaaccac 6000 ccctccagaa cgtgtaccaa ctcgttcacg aggtgattga acgggtcgag ctccacgaga 6060 aggtgtcgag ccacccgatt ttcaaacaga tatcaattcc ttcccaaaag tctgtgttgt 6120 acttcctcat tgagaaaggc caacacgaag cagcaattga attctttgag ggaatggtgc 6180 atgactccat caaggaagag ctccggcccc tcatccaaca gacctcattt gtgaaacgcg 6240 cttttaagcg cctgaaggaa aactttgaga ctgttgccct gtgtttgact cttttggcaa 6300 acatagtgat catgatccgc gagactcgca agagacaaca gatggtggac gatgcagtga 6360 atgactacat tgagaaggca aacatcacca cagatgacaa gactcttgac gaggcggaaa 6420 agaaccctct agagaccagc ggtgccagca ctattggttt cagagagaga actctcccgg 6480 ggcacaaggc gagcgatgac gtgagctccg agcccgccaa acccgtggag gaccgaccac 6540 aagctgaagg accttacgag ggaccggtga agaagcctgt cgctttgaaa gtgaaagcta 6600 agaacttgat tgtcactgag gggccatatg aaggaccagt gaagaaacct gtcgctttga 6660 aagtgaaagc aaaagccccg attgtcactg aaggacccta cgagggaccg gtgaagaagc 6720 ctgtcgcttt gaaagtgaaa gccaagaact tgattgtcac tgagagtggt gccccaccga 6780 ccgacttgca gaagatggtc atgggcaaca ctaagcctgt tgagctcatc ctcgacggga 6840 agacggtagc catctgctgt gctaccggag tgtttggcac tgcctacctc gtacctcgtc 6900 acctcttcgc ggagaagtac gacaagataa tgttggacgg tagagccatg acagacagtg 6960 actacagagt gtttgagttt gagattaaag taaaaggaca ggacatgctc tcagacgctg 7020 cactcatggt gcttcaccgt gggaaccgcg tgagagacat cacgaaacat tttcgtgaca 7080 cagcaagaat gaagaaaggc acccccgttg tcggtgtgat caacaacgcc gacgttggga 7140 gactgatttt ctctggagag gcccttacct acaaagacat tgtagtgacc atggatggag 7200 acaccatgcc gggcctgttt gcctacagag ccgccaccaa ggctggttac tgcgggggag 7260 ccgttctcgc caaggacgga gccgacacat tcatcgttgg cacccactcc gcaggtggta 7320 acggagttgg atactgctcg tgcgtgtcca ggtccatgct cctgaaaatg aaggcacaca 7380 ttgaccctga accacaccac gaggggttga ttgttgatac cagagatgtg gaagagcgcg 7440 tgcatgtcat gcgtaaaacc aagcttgcac ccaccgtggc acacggtgtg tttaaccctg 7500 aatttggtcc cgctgccttg tccaaacaagg acccgcggct gaacgaaggg gttgtcctcg 7560 atgaagtcat cttctccaaa cacaagggag acacgaaaat gtctgaggag gacaaagcgc 7620 tgttccgccg ctgcgctgcc gactacgcgt cgcacttgca cagcgtgctg gggacggcaa 7680 atgccccatt gagcatctat gaggccatca aaggcgtcga cgggctcgat gccatggagc 7740 cggacaccgc gcccggcctc ccctgggccc tccaggggaa acgccgtggt gcgttgattg 7800 acttcgagaa cggcacggtc ggacccgaag tcgaggctgc cctaaagctc atggagaaaa 7860 gagagtacaa atttgcttgc cagaccttcc tgaaagacga gattcgtccg atggaaaaag 7920 tacgtgctgg caagactcgc attgtcgacg ttttgcccgt ggaacacatt ctttacacca 7980 ggatgatgat tggcagattc tgtgctcaaa tgcacacaaa caatggaccg cagattggct 8040 cagcggtcgg ttgcaatcct gatgttgatt ggcaaagatt tggcacacat tttgctcagt 8100 acagaaacgt gtgggatgtg gactattcgg cctttgatgc taaccactgc agtgacgcaa 8160 tgaacatcat gtttgaggag gtatttcgca cagacttcgg tttccaccca aatgctgagt 8220 ggattctgaa gactcttgtg aacacggagc acgcctatga gaacaaacgt atcactgttg 8280 agggcgggat gccgtctggc tgttccgcga caagcatcat caacacaatt ttgaacaaca 8340 tttatgtgct ctacgctctt cgtagacact atgagggagt tgagctggac acctacacca 8400 tgatctccta cggagatgac atcgtggttg caagtgacta cgatctggat tttgaggctc 8460 tcaaacccca cttcaaatct cttggtcaaa ccatcactcc agctgacaaa agcgacaaag 8520 gttttgttct tggtcactcc attaccgatg tcactttcct caaaagacac ttccacatgg 8580 actatggaac tgggttttac aaacctgtga tggcctcaaa gaccctcgag gccattctct 8640 cctttgcacg ccgtgggacc atacaggaga agttgatctc cgtggcagga ctcgccgtcc 8700 actcaggacc tgacgagtac cggcgtctct ttgagccctt ccagggtctc ttcgagattc 8760 caagctacag atcactttac ctgcgttggg tgaacgccgt gtgcggtgac gcataatccc 8820 tcagatgtca caattggcag aaagactctg aggcgagcga cgccgtagga gtgaaaagcc 8880 cgaaagggct tttcccgctt cctattccaa aaaaaaaaaaa aaaaaactag ttctagagcg 8940 gccgccaccg cggtggagct ccagcttttg ttccctttag tgagggttaa ttgcgcgctt 9000 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 9060 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 9120 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 9180 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 9240 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 9300 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 9360 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 9420 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 9480 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 9540 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 9600 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 9660 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 9720 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 9780 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 9840 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 9900 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 9960 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 10020 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 10080 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 10140 ctaaagtata tatgagtaaa cttggtctga cagttaaccaa tgcttaatca gtgaggcacc 10200 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat 10260 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc 10320 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag 10380 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag 10440 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt 10500 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg 10560 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt 10620 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc 10680 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc 10740 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacggggataa 10800 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg 10860 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc 10920 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag 10980 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt 11040 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt 11100 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc 11160 ac 11162 <210> 9 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> G7E epitope amino acids <400> 9 Ser Gly Arg Val Arg Gly Asp Leu Gly Gln Leu Ala Ala Arg Val Ala 1 5 10 15 Ala Gln Leu Pro Ala 20 <210> 10 < 211> 21 <212> PRT <213> Artificial Sequence <220> <223> G2E epitope amino acids <400> 10 Gln Asn Arg Arg Gly Asp Ser Gly Pro Leu Val Val Lys Pro Ala Thr 1 5 10 15 Gln Leu Pro Ala Ser 20 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 accatggatg gagacaccat g 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cactacaatg tctttgtagg ta 22 <210> 13 <211> 40 <212> DNA <213 > Artificial Sequence <220> <223> primer <400> 13 aggaccgacc acaagctgaa ggaccttacg agggaccggt 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tcggtcggtg gggcaccact ctcagtgaca atcaagttct 40 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 accactacca ccggggag 18 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223 > primer <400> 16 ttgttttgca ggtgcaatga t 21 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 cttctaaatt ttgacctgct caaattggcg gg 32 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 ttgcgcacgg gggtcaat 18 <210> 19 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 tcgcggcgcg agtcgctgct caactccctg ccgcgagggt cgccgctca 49 <210> 20 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 gctgtcccag gtcaccccgt acacgcccac tcgcgagagg ccctaggtc 49 <210> 21 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tggtgaaacc cgccacacag ctccctgcct ccgcgagggt cgccgctcag 50 <210> 22 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> primer<400> 22 cgagaggacc cgagtcacct cgccggtttt gcgcgagagg ccctaggtc 49

Claims (7)

A recombinant foot-and-mouth disease virus having sequences 646 to 8926 of sequence number 7 or sequences 642 to 8926 of sequence number 8. A recombinant plasmid having sequence number 7 or sequence number 8 for producing a recombinant foot-and-mouth disease virus of claim 1. Foot-and-mouth disease A 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 A 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 cloven-hoofed animals other than humans using the foot-and-mouth disease diagnostic kit of Article 5. A method for preventing or treating foot-and-mouth disease in a clonal animal other than a human, using a vaccine composition comprising the recombinant foot-and-mouth disease virus of claim 1 or the recombinant foot-and-mouth disease virus antigen of claim 3.