KR102623115B1 - Novel foot-and-mouth disease Asia1 recombinant virus and foot-and-mouth disease vaccine composition comprising the same - Google Patents

Abstract

The present invention relates to a foot-and-mouth disease recombinant virus and a foot-and-mouth disease vaccine composition containing the same. Specifically, it relates to a novel foot-and-mouth disease Asia1-type recombinant virus with excellent protective activity against Asia1-type virus and a foot-and-mouth disease vaccine composition containing the virus.
In addition, the present invention can provide a recombinant foot-and-mouth disease virus with strong protective ability against foot-and-mouth disease Asia1 type virus, a vaccine composition for preventing foot-and-mouth disease containing the same as an active ingredient, and a method for producing the same.

Description

Novel foot-and-mouth disease Asia1 recombinant virus and foot-and-mouth disease vaccine composition comprising the same}

The present invention relates to a foot-and-mouth disease recombinant virus and a foot-and-mouth disease vaccine composition containing the same. Specifically, it relates to a novel foot-and-mouth disease Asia1-type recombinant virus with excellent protective activity against Asia1-type virus and a foot-and-mouth disease vaccine composition containing the virus.

Foot-and-mouth disease (FMD) is a disease that mainly infects cloven-hoofed animals (artiodactyls) such as cattle, pigs, sheep, goats, deer, and wild ruminants. It is highly contagious, and is clinically diagnosed in the lips. It is a disease that causes blisters to form on the tongue, gums, nose, etc., body temperature rises rapidly, appetite decreases, and in young animals, myocarditis occurs and death occurs. There are seven serotypes of foot-and-mouth disease virus, and each virus has subtypes, so even with the same serotype, protection may not be possible depending on the virus type. In the case of Asia1 serotype, most cases occur only in Asia, so there is only one regional type, ASIA. Among these, they are divided into genetic groups from group I to group IX.

Asia1 serotype was first reported in South Asia and Southeast Asia in 1951, and its incidence increased in East Asia from 2004 to 2007. Asia1 serotype was identified in Pakistan in 1954, and in India, Asia1 serotype is the most frequently occurring virus serotype after type O virus. The virus that emerged in China and Russia was confirmed to be similar to the virus that emerged in India in 1980/81. In East Asia, group V and group IV occurred frequently and became prevalent. The Indian-origin virus that occurred between 2001 and 2004 was identified as group III. Group VIII is a virus that has occurred in India since 2005. In 2018, a new group of viruses classified as group IX was identified in Bangladesh, and viruses classified as new virus strains are continuously occurring.

In addition to the O and A serotypes that occur globally, the Asia1 serotype belonging to pool 1 is included, and these viruses have recently become prevalent in the Asia region, so advance preparation for the outbreak of this type of foot-and-mouth disease is necessary. There is a need to develop vaccine compositions.

Public Patent Publication No. 10-2011-0088526 Public Patent Publication No. 10-2019-0082081

Lee SY, Lee YJ, Kim RH, Park JN, Park ME, Ko MK, Choi JH, Chu JQ, Lee KN, Kim SM, Tark D, Lee HS, Ko YJ, Seo MG, Park JW, Kim B, Lee MH , Lee JS, Park JH. 2017. Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses. J Virol 91. Ito N, Takayama-Ito M, Yamada K, Hosokawa J, Sugiyama M, Minamoto N. 2003. Improved recovery of rabies virus from cloned cDNA using a vaccinia virus-free reverse genetics system. Microbiol Immunol 47:613-7.

In order to solve the above problems, the present invention seeks to provide a recombinant foot-and-mouth disease virus with excellent protective ability against foot-and-mouth disease virus, especially Asia 1 type, and a vaccine composition containing the virus.

It is also intended to provide a method for producing the recombinant virus and vaccine composition.

In order to achieve the above object, the present invention provides a novel foot-and-mouth disease recombinant virus produced based on foot-and-mouth disease virus Asia1 type.

The Asia1 type is not limited, but may be Asia1/Shamir/89 in an embodiment of the present invention.

The recombinant virus according to the present invention consists of the sequence of SEQ ID NO: 12. After cell culture with virus ZZR 5 times, BHK21 5 times, and BHK21S suspension cells 6 times, the difference from SEQ ID NO: 12 is that the virus has natural mutations of C3586T, G3594A, and A3770C based on plasmid SEQ ID NO: 13, and the amino acids are VP1-Q110R, The D169N mutation was confirmed.

Additionally, the present invention provides a recombinant plasmid used for producing the recombinant virus, and the recombinant plasmid consists of the sequence of SEQ ID NO: 13.

Additionally, the present invention can provide a foot-and-mouth disease virus antigen obtained by purifying and isolating the recombinant foot-and-mouth disease virus. It has excellent protective properties against the recombinant foot-and-mouth disease virus or the foot-and-mouth disease virus containing the antigen isolated and purified from the recombinant foot-and-mouth disease virus, especially Asia1 type.

In addition, the present invention can provide a vaccine composition containing the recombinant foot-and-mouth disease virus or foot-and-mouth disease virus antigen, and the vaccine composition may be specifically for preventing, improving, or treating foot-and-mouth disease, but is not limited thereto.

The present invention provides a method for preventing, improving, or treating foot-and-mouth disease using the vaccine composition.

As used herein, the term “plasmid” refers to a DNA preparation containing a DNA sequence operably linked to a suitable control sequence 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, “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. A typical plasmid vector that can be used for this purpose includes (a) an origin of replication that allows efficient replication to contain hundreds of plasmid vectors per host cell, and (b) a replication origin that allows host cells transformed with the plasmid vector to be selected. It has a structure that includes a selection marker and (c) a restriction enzyme cut site into which a foreign DNA fragment can be inserted. Even if an appropriate restriction enzyme cut site does not exist, the vector and 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 produced by conventional genetic engineering and 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 of the present invention can be used in canines, felines, boars, bovines, deers, giraffes, peccaries, camelids, hippopotamuses, equines, tapirs, rhinoceroses, weasels, lagomorphs, It may be derived from one or more types of cells selected from the group consisting of rodent and primate cells, preferably goat tongue cells (ZZ-R), hamster kidney cells (BHK-21), black goat kidney cells (BGK), and pig cells. One or more types selected from the group consisting of kidney cells (IBRS-2) and bovine kidney cells (LFBK) can be used.

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

The vaccine composition can be administered to artiodactyls such as cattle, pigs, sheep, goats, deer, and wild ruminants, excluding humans.

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

Additionally, the vaccine composition may be administered (or injected) to an individual 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.

Additionally, the present invention can provide a method for producing the above recombinant virus, and the production method includes the following steps;

(a) preparing a foot-and-mouth disease Asia1 type recombinant plasmid;

(b) isolating and recovering the recombinant foot-and-mouth disease virus from the recombinant plasmid obtained in step (a).

The present invention can provide a recombinant foot-and-mouth disease virus with strong protective ability against foot-and-mouth disease Asia1 type virus and a vaccine composition for preventing foot-and-mouth disease containing the same as an active ingredient.

Additionally, a method for producing the recombinant virus and vaccine composition may be provided.

Figures 1A to 1D show the characteristics of chimeric foot-and-mouth disease viruses by Asia1 Shamir, As-G-VIII, As-Sham-G-VIII EPI, and As-Sham-G-VIII VP1 (Figure 1A shows the characteristics of the new Asia1 virus A schematic diagram of a chimeric virus expressing a protective antigen; Figure 1b shows the results of recombinant FMDV differentiation through non-structural proteins using a rapid diagnostic kit (PBM, USA); Figure 1c shows an image of chimeric virus particles through an electron microscope) , Figure 1d shows a schematic diagram of the recombinant plasmid that serves as the basis for virus production).
Figure 2a shows the modified sequence for the 3B region (3B1B2B3 to 3B3B3B3[=3B333]).
Figure 2b shows the sequence modified with the corrected C142T base sequence for the 3C region (replacement of the start site of SEQ ID NO: 8 (TGC->ACC)).
Figures 3a to 3d show the genetic differences between Asia1 serotype viruses and vaccine viruses (A. Genetic similarity between Asia1 lineage, GI to G-VIII lineage and Asia1 Shamir vaccine lineage, B. Asia1 lineage, GI to G Genetic similarity between -VIII lineage and Asia1 IND115 (G-VIII) lineage, C. Asia1 phylogenetic tree strains, GI to G-VIII lineage and vaccine strain, and amino acid mutations in the VP1 region of D. Asia1 strain. Indicated. Boxes are the BC and GH loops and hypervariable region of the VP1 region). *GenBank Accession; IND/8/79 (EU553910), IND115/2012(KT153359), IND 2013(IND156(335)/2013, KT153367) BD_SI_2_2013(KR869774), BAL_PAK_iso-1_2011(JX435110), TUR/13/20 13(KM268898), Asia1 /Shamir/89 (MF063053), PAK/8/2008 (KY091304), AFG/1/2001 (DQ121109), IRN/10/2004 (DQ121119), IND762-03 (DQ101240), VN/LC04/2005 (GU125646) , HKN/19/74 FJ785230, IND/18/80 DQ121116, As/MOG/2005 (EF614458), IND/14/95 (AF390678), NKR/2/2007 (FJ785259).
Figure 4 shows the survival results of immunized C57BL/6 mice vaccinated with Asia1 Shamir wild-type virus 7 days after vaccination at a diluted antigen dose (Asia1-Shamir-R) (A. Asia1 Shamir 100 LD ₅₀ Survival rate of the 7 dpv group after vaccination, and weight change of the 7 dpv group after B. Asia1 Shamir vaccination).
Figures 5a and 5b show the immunogenicity and protection results of mice vaccinated with the antigen of the recombinant vaccine virus (A. Shamir-R, Shamir-G at 28 dpv after inoculation with Asia1 Shamir virus of 100 LD ₅₀ -VIII-EPI and Shamir-G-VIII-VP1 survival rate of immunized group, B. Body weight change of immunized group after Asia1 Shamir vaccination, C. at 28 dpv after vaccination with 100 LD ₅₀ of Asia1 G-VIII virus Survival rate of Shamir-R, Shamir-G-VIII-EPI and Shamir-G-VIII-VP1 immunized groups, D. Body weight change of immunized group after Asia1 G-VIII vaccination, E. Vaccinated mice (n = 8) Antigenic inhibition efficiency (ELISA antibody level) of antibodies in Asia1 type SP-ELISA, F. Asia1 Shamir virus as measured by virus neutralization test in Asia1 Shamir-R, G-VIII EPI and G-VIII VP1 immunized mice. Immune response to G. Asia1 Shamir-R, G-VIII EPI, and G-VIII VP1 immunized mice. Immune response to Asia1 G-VIII virus as measured by virus neutralization test.
Figures 6A and 6B show the results of cross-virus neutralizing antibody tests against various Asia1 gene lineage viruses in pigs immunized with Asia1-Shamir-R (viruses for pigs immunized with Asia1-Shamir-R (n = 5)). Neutralizing antibodies: A. Asia1 Shamir, B. Asia1-Shamir-R, C. O1ma LC-04 (G-IV), D. Asia1 MOG (GV), E. Tur/13/2013 (G-VII), F .Asia1 G VIII-EPI, G. Asia 1 G VIII-VPI, H. AG VIII).

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

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

[Experimental materials and methods]

1. 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). % 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. The target viral sequences selected for this experiment were Asia1/Shamir/89 (Genbank No. JF739177.1), IND115 (217)/2012 (Genbank No. KT153359.1), and BD_SI_2_2013 VP1 (Genbank. KR869774.1).

2. Site-directed mutagenesis and subcloning

For this experiment, the P1 region (SEQ ID NO: 2) of Asia1/Shamir/89 (Genbank No. JF739177.1) (SEQ ID NO: 1) IND115 (217)/2012 (Genbank No. KT153359.1) and BD_SI_2_2013 VP1 (Genbank KR869774 .1) was replaced with the VP1 region (SEQ ID NO: 3), and IND115 (217)/2012 (Genbank No. KT153359) of gene group VIII ((IND115 (217)/2012 and BD_SI_2_2013 strains were identical in the VP1 amino acid sequence). 1), the VP1 sequence from 140 to 160 (SEQ ID NO: 4, its amino acid sequence is SEQ ID NO: 5) was inserted into the 5th amino acid region from the cell-binding receptor (RGD) inside VP1.

In order to replace the 3B1 and 3B2 regions with two 3B3s, a full genome cDNA vector with both 3B1 and 3B2 deleted was used, and the synthesized 3B3 dimer gene was used as a PCR template at the 3B1 and 3B2 regions to apply specific primers (5 The 3B3 dimer gene amplified with '-AGGAACAACCACAGGCTGAAGGACCTTACGAGGGACCGGT-3' (SEQ ID NO: 6) and 5'-TCGGTCGGTGGGGCACCACTCTCAGTGACAATCAAGTTCT-3' (SEQ ID NO: 7) was inserted. Gene sequence analysis of the constructed recombinant plasmid confirmed the 3B amino acid substitution of As-Sham-GVIII EPI and As-Sham-GVIII EPI viruses (SEQ ID NO: 8, the amino acid sequence of which is SEQ ID NO: 9) (FIG. 2a).

The virus was genetically modified to create a new, less pathogenic virus by transfection into BHK T7-9 cells. The plasmid into which the entire foot-and-mouth disease virus gene was inserted was used as a PCR template for amino acid substitution with the 3C amino acid C142T (TGC -> ACC). The entire plasmid gene was amplified using (5'-ACCATGGATGGAGACACCATG-3' (SEQ ID NO. 10) and 5'-CACTACAATGTCCTTGTAGGTG-3' (SEQ ID NO. 11), then self-ligated, and the constructed recombinant plasmid was sequenced to determine 3C amino acids. The substitution was confirmed. As-Sham-GVIII EPI and As-Sham-GVIII EPI viruses were produced by transfection into BHK T7-9 cells (Figure 2b).

3. Virus recovery and cell culture

The recovered virus was obtained using the same procedure as in the previous study (see Non-Patent Document 1). Specifically, the recombinant cDNA plasmid was linearized through restriction enzyme SpeI (NEB, Beverly, USA) treatment, and the infectious cDNA was grown in BHK-T7-9 cells (a cell line in which T7 RNA polymerase is expressed) (see Non-Patent Document 2). After incubation at 37°C for 48 h, the virus was harvested through three freeze-thaw cycles and the chimeric virus was used to infect fresh ZZ-R cells. For differentiation from wild-type FMDV and recombinant viruses, structural and non-structural proteins were identified using the FMDV Rapid Kit (Princeton Bio Meditech Corporation, USA). The sequence of the recombinant virus finally obtained using the above plasmid is shown as SEQ ID NO: 12.

4. Virus purification for vaccine antigen

To produce antigens for vaccine production, the recombinant virus was cloned using BHK21 or BHK21-suspension cells, which are cells for FMDV production. Sixteen hours after infection, viruses grown in BHK 21 suspension cells were harvested by freezing and thawing when complete CPE was observed. The virus was inactivated with binary ethylene imine (BEI) and 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-NaCl (TN) 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 a SW41 rotor at 4°C, and the antigen amount was determined by spectrophotometric analysis at 259 nm. Purified antigens were separately adsorbed onto carbon-coated copper grids and visualized by transmission electron microscopy (Hitachi H7100FA, Tokyo, Japan).

5. Preparation of experimental vaccine

The vaccine was manufactured using the method used in previous studies (see Non-Patent Document 1). That is, purified 146S of Asia1 Shamir-R (SEQ ID NO: 12), As-Sham-G VIII EPⅠ (SEQ ID NO: 14), and As-Sham-G VIII VP1 (SEQ ID NO: 15), which are recombinant viruses obtained in step 3 above. 15 μg of antigen (1 batch) 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 into this mixture to form a water-in-oil-in-water solution. ) The vaccine was manufactured.

6. Genetic variation among FMD Asia 1 type virus

Genetic similarity analysis of the phylogenetic tree was performed using MEGA software (ver. 6.0) with BioEdit and neighbor-joining method for Windows 95/98/NT2K/XP/7. The proportion of replicates (1000 replicates) in the tree in which related taxa clustered together in the bootstrap test is shown next to the branches.

7. Virus challenge after vaccination in adult C57BL/6 mice

In this experiment, 7-week-old C57BL/6 female mice supplied by KOSA-BIO (Republic of Korea) were used. Animals were housed at APQA and used with approval from the Animal Care and Use Committee. Antigens (2.0, 1.5, 0.5, 0.375, 0.125, 0.093, 0.031, 0.023 and 0.016) mixed with ISA 206 (Seppic, France) in dose groups (dose of vaccination for pigs or cattle (15 μg/dose)) and 10% aluminum hydroxide gel adjuvant was administered. Serum was collected up to 4 weeks after vaccination (0, 7, 14, 21, and 28 dpv). 28 days after vaccination (dpv), all mice were intraperitoneally administered 1x10^5 TCID50 and then challenged with Asia1 Shamir-WT virus and observed for 7 days.

8. ELISA for the detection of structural protein antibodies

Structural proteins of FMDV in serum were detected using PrioCheck FMDV Asia1 (Prionics, Switzerland). Animals were considered to have exhibited an immune response if the sample reflected a percent inhibition (PI) value of ≥50%.

9. Test of the immunogenicity of the test vaccine in pigs

Pigs (n=5) were inoculated with 15 μg/ml antigen mixed with ISA 206, saponin, and aluminum hydroxide gel adjuvant. Serum was collected up to 4 weeks post-vaccination (7, 14, 21, and 28 dpv). Animals were housed at APQA and used with approval from the Animal Care and Use Committee.

10. Virus neutralization test in mice and pigs

After vaccination, serum samples were collected from pigs and mice and heat-inactivated at 56°C for 30 minutes. After the test serum was incubated with FMDV 100 TCID ₅₀ for 1 hour, LFBK cells were added to the plate and cultured for 3 days. CPE was determined to determine titer, which was calculated as log10 of the reciprocal serum dilution to neutralize 100 TCID ₅₀ of the virus. FMDV Asia1 Shamir and Asia1 Shamir-R were used for VNT in mouse serum. FMDV Asia1 Shamir-R, Asia Shamir-WT, O1ma LC04 P1, Asia1 MOG, Asia1 Turkey/13/2013, As-G VIII, As-Sham-G VIII EPⅠ and As-Sham-G VIII VP1 VNT in pig serum was used in

[Experiment result]

1. Characteristics of the recombinant FMDV

The whole genome cDNA of Asia1 Shamir virus was cloned under the binding site of T7 RNA polymerase. The epitope (residues 140–160) of Asia1 G-VIII against Asia1 Shamir or VP1 was added or replaced for the preparation of new vaccines. Viruses were recovered from cells transfected with cDNA. Asia1 IND115/2012 (G-VIII) P1-phenotype virus was used as challenge virus against G-VIII lineage virus for vaccine evaluation (Figure 1a). 3B1B2 in the viral genome was replaced by 3B3B3, which could be distinguished from the vaccine virus and the wild-type strain of Asia1 Shamir using a simple antigen diagnostic kit (lateral flow device; LFD) that allows detection of NSP without reaction by the replacement site. Additionally, the vaccine virus in this experiment confirmed the expected size of FMDV at 25 nm through electron microscopy (Figure 1c).

2. Genetic differences between Asia1 viruses

The difference in genetic homology with Shamir virus, the standard vaccine strain, showed 83-91% homology for nucleotides and 88-95% for amino acids for each genotype. The genotype with the highest amino acid similarity was Asia1/IRN/10/2004, G-II. The amino acid similarity with the G-VIII virus was not very high at 88-93%. The genotype with the lowest amino acid identity rate was 88% for IND 2013, a G-VIII virus. The difference in genetic homology with the IND 115/2012 virus, a G-VIII virus, was 84-99% for nucleotides and 88-100% for amino acids for each genotype. The genotypes with the highest amino acid similarity to G-VIII were G-II, Asia1/IRN/10/2004, and G-VI, Asia1 IND 14/95. The similarity with the G-VII virus is 88-89%, followed by G-I AFG/1/2001 at 89%, G-IV LC04 at 90%, and G-V with AsMOG and NKR/2/2007 at 90%. The similarity was relatively low. In VP1, it was confirmed that residues 44-48, 96-101, and 137-156 were significantly changed (Figures 3a to 3d).

3. Asia1 Shamir virus challenge test in mice after Shamir-R vaccination)

When mice immunized with 0.1 ml of 2.0-0.016 μg of 146S particles were inoculated at 7 dp with the recombinant virus according to the invention, all mice immunized with 0.016 μg of final dilution of antigen survived. Therefore, the protection rate of the recombinant viral vaccine according to the present invention against Asia1 Shamir was determined to be > 38.1 mouse protective capacity PD50 (1.5 μg/dose for mouse) (Figure 4, A and B). The effect on body weight loss was not observed in mice (Figure 4B). In the recombinant virus immunization group according to the present invention containing two types of newly created viral antigens, both Shamir and G-VIII viruses were attacked one week after immunization with 1.5 μg, and protective ability was confirmed (FIGS. 5A and 5B). As a result of the immunogenicity test in mice, similar results were confirmed. A higher level of antibody reactivity was confirmed in the neutralization test results for homologous viruses among each virus (Figures 5a and 5b).

4. Comparison of antibodies against several virus strains after immunization in pigs

SP-ELISA and antibody titers for virus neutralization were compared after vaccination in pigs (Figures 5A and 5B). A virus neutralization test was performed to compare the titers of antibodies against the Asia1 strain in pigs immunized with the experimental vaccine (Figures 6A and 6B). Pigs developed significantly higher antibody titers (>1:100) 3 weeks after vaccination (Figure 6A, A and B). After the first vaccination, immunogenicity tended to gradually increase up to 3 to 4 weeks. In the neutralization test, titers of neutralizing antibodies against Asia1 MOG or LC04 were found to be barely high, with the highest antibody reactivity against Asia1 Shamir. The titers of neutralizing antibodies against Asia1 G-VIII, Shamir-GVIII EPI, Shamir-GVIII VP1 and Asia1 Tur13/2013 virus strains were determined to be high (Figures 6A and 6B).

5. Comprehensive results

As a result of the neutralization test after vaccination with the test vaccine against the recombinant virus according to the present invention, the neutralizing antibodies against the recombinant virus according to the present invention, Turkey/13/2013, and the neutralizing antibody against the homologous virus, the recombinant virus according to the present invention, were compared. Antibody titers were similarly high in most groups. In addition, a similarly high level of neutralizing antibody formation was confirmed for the G-VIII virus and the newly created fusion vaccine strains Shamir-G-VIII-Epi and Shamir -G-VIII-VP1 viruses. Therefore, it was confirmed that the antigenicity of not only the recombinant virus of the present invention, but also Shamir-G-VIII-Epi and Shamir-G-VIII-VP1, which were additionally studied, showed a similar tendency to that of the recombinant virus of the present invention. Therefore, since there was no difference in antigenicity, when comprehensively judging the recombinant virus of the present invention, which was selected as a representative of the three viruses and evaluated in pigs, it was found that the antigen matched with the currently used viruses G-VII and G-VIII viruses. It was assessed that there was a difference in antigenicity from G-IV and G-V, which had relatively low levels of neutralizing antibodies.

In conclusion, the Shamir-G-VIII-Epi and Shamir-G-VIII-VP1 vaccine strains containing the recombinant virus according to the present invention are expected to be able to protect against Shamir family pseudoviruses and G-VII and G-VIII in pigs. It is judged. In addition, two additional vaccines developed specifically for G-VIII are Shamir-G-VIII-Epi and Shamir-G-VIII-VP1, which are similar to or can be used as a broader vaccine than Shamir, which is widely used as an international vaccine strain. It is judged that

<110> Animal and Plant Quarantine Agency <120> Novel foot-and-mouth disease Asia1 recombinant virus and foot-and-mouth disease vaccine composition comprising the same <130> 21-11998 <160> 15 <170> KoPatentIn 3.0 <210 > 1 <211> 8196 <212> DNA <213> Artificial Sequence <220> <223> Asia1/Shamir/89 (Genbank No. JF739177.1) <400> 1 ttgaaagggg gcgctagggt ctcaccccta gcacgccaac gacagctcct gcattgcact 60 ccacacttac gtctgt gcac atgcgggaac cgctggacta tcgttcaccc acctacggct 120 ggactcacgg caccgcgtgg ccatgctagc tggattgtgc ggacgaacgc catttgcgca 180 tctcgcgtgg ctggttaata ctcttaccac tttccgccta cctggtcgtt agcgctgtct 240 tgggcactcc cgttgggggc cgttcgatgc t ccacggtct cccccgtgtg acaaactacg 300 gtgttgggac cgctttgtgc gagccgctcg cctggtgtgc ttcggctgtc actcgaagcc 360 cgcctttcac cccccccccc ctaagtttta ccgtcatccc gacgttaaag ggaaccaacc 420 acaagcttgg aaccgtct cg cccgacgtga aaggggctgt aaccacaagc ttgcaccgcc 480 tttcccggcg tttaacggga tgtaaccaca agataaacct tcgcccggaa gtaaaacgac 540 aacttcacac acttttgccc gtttttatga gaaatgggac gtctgcgcac gaaacgcgcc 600 gtcgcttgag gaatacctgt acaaacacga ctcacgcagg tctccacaac tgacacaaac 660 cgtgcaactt gaaactccgc ctggtctttc caggtctaga gg ggtgacac tttgtactgt 720 gcttgactcc acgctcggtc cactagcgag tgttagtaac agcactgttg cttcgtagcg 780 gagcatggtg gccgtgggaa cccctccttg gcgacaagga cccacggggc cgaaagccac 840 gtccttgtgg acccaccatg tgtgcaaccc cagcacggca gctctactgt gaaaaccact 900 ttaaggtgac actgatactg gtactcagac actggtgaca ggctaaggat gcccttcagg 960 taccccgagg taacacgcga cactcgggat ctgagaaggg gactgagact tctttaaaag 1020 tgcccagttt aaaaagcttc tatgcctgaa taggcgaccg gaggccggcg cctttctcct 1080 taactaccac ttcttaatga acacaactga ctgttttatc gctctgctgc acgccctcag 11 40 agagatcaaa acgctgtttc tgtcacggac ccaagggaaa atggaattca cactctacaa 1200 cggtgagaag aagacctttt actccaggcc caacaaccac gacaactgtt ggttgaacac 1260 catcctccag ctgtttaggt acgttgatga gcctttcttt gactgggttt atgact cgcc 1320 cgagaacctc acccttgctg caattgaaca gttggaagag gttaccggtc ttgagctgca 1380 cgaaggcggg ccacccgctc tcgtcatttg gaacatcaaa cacttgctcc acactggaat 1440 cggtactgct tcgcgaccca gcgaggtgtg tatggtggac ggcacggaca tgtgtttggc 1500 tgacttccac gccggcattt tcctgaaagg tcaggaacat gccgtgtttg cctgtgt cac 1560 ctccaacggg tggtatgcga ttgatgatga ggacttttac ccttggacgc cggacccgtc 1620 cgacgttctg gtgtttgttc cgtacgatca ggaaccgctc aacggggaat ggaaagcaaa 1680 ggttcagaaa cgacttaagg gagccggtca atccagtcc g gcaaccgggt cacagaacca 1740 atctggcaac actggaagca tcattaacaa ctactacatg caacagtacc agaattccat 1800 ggacacacag cttggtgaca acgctattag cggaggttcc aacgaaggtt ccacggatac 1860 cacttccaca cacacaaaca acacccaaaa caacgactgg ttctcgcgcc tggctagctc 1920 tgcattcagt ggtctctttg gtgcactttt ggctgacaag aagacagaag agacaactct 1980 gcttgaagac cg cattctca ccaccaggaa cggccacaca acatcgacga cacagtcgag 2040 cgttggcgta acatacggtt acgctgtggc cgaggacgcg gtgtctggac ccaatacctc 2100 gggtctagag actcgtgttc aacaggcaga acggtttttc aagaaacacc tgtttgactg 21 60 gacaccgaac ttggcatttg gacactgtta ctacctggaa cttcccactg aacacaaagg 2220 cgtgtacggc agtctcatgg gctcgtacgc ctacatgaga aatggatggg acatagaggt 2280 gactgctgtt ggaaaccaat tcaacggtgg ttgtctcctt gtcgcgctcg tgccagagct 2340 gaaggaactc gacacgcgac agaagtacca gctgaccctc tttccccacc agttcatcaa 2400 cccacgcacc aacatgacgg cc cacatcaa cgtgccgtac gtgggtatca acaggtacga 2460 ccagtacgcc ctccacaagc cgtggacgct tgttgtgatg gtggtagccc cactcaccgt 2520 caaaactggt ggttctgaac agatcaaggt ttacatgaat gcagcgccaa cctacgtgca 2580 tgtggc ggga gagctgccct cgaaagaggg aatagttccc gtcgcgtgtg cggacggtta 2640 cggcaacatg gtgaccacgg acccgaagac ggccgatcca gtttacggga aagtgttcaa 2700 cccccccagg acaaacctcc ctgggcgctt cacgaacttc cttgatgttg cggaggcatg 2760 tccaactttc ctccgctttg gagaagtacc atttgtgaag acggtgaact ctggtgaccg 2820 cttgctggcc aagttcgac g tgtccctcgc tgcagggcac atgtccaaca cctacttggc 2880 tggcctggcg cagtactaca cacagtacag cggcaccatg aacgtccact tcatgttcac 2940 cgggcccacg gatgctaaag cccgatacat ggtggcttat gtcccccctg gcatgacacc 3000 gcccacggac c ctgagcacg ccgcacactg cattcactct gagtgggata ctggtcttaa 3060 ctctaagttt accttttcca taccttacct ctctgctgct gactatgcct acactgcttc 3120 tgacgtggcg gagaccacga gtgtgcaggg atgggtgtgt atctatcaga tcacccacgg 3180 caaggctgag ggagacgcac tggtcgtttc tgtcagcgcc ggcaaagact ttgagtttcg 3240 cttgcctgtt gacgcacgcc ag caaaccac caccactggc gaatcagcag atccagtcac 3300 aaccacggtt gagaactatg gaggagagac tcagacagcc agacggcttc acactgacgt 3360 cgccttcatt cttgacaggt ttgtgaaact cactgctccc aagaacatcc aaaccctcga 3420 tctcatgcag atcccctcac ac acgctggt tggagcacta cttcgttctg cgacgtacta 3480 cttctcagac ctggaggtcg cgcttgtcca cacaggcccg gtcacctggg tgcccaacgg 3540 cgcgcccaag gatgctctaa acaaccagac caacccaact gcctatcaga agcaacccat 3600 cacccgcctg gcactcccct acaccgcccc ccatcgtgtg ctggcaacag tgtacaacgg 3660 gaagacggcg tacggggaaa cgacctcaag gcgcggcgac atggcggccc tc gcacaaag 3720 gttgagcgct cggctgccca cctccttcaa ctacggcgcc gtgaaggccg acaccatcac 3780 tgagcttttg atccgcatga agcgcgcgga gacatattgc cctaggcctt tactagccct 3840 tgacaccact caggaccgcc gcaaacagga gatcattgca c ctgagaagc aggttttgaa 3900 ctttgaccta ctcaagttgg caggagacgt tgagtccaac cctgggccct tcttcttctc 3960 cgacgttagg tcgaacttct ccaaactggt cgagaccatc aaccagatgc aggaggacat 4020 gtcaacaaag cacggacccg acttcaaccg gttggtttcc gcgtttgagg aattggccac 4080 aggagtaaag gccatcagga acggtctcga tgaggccaag ccctggtaca agctcatcaa 41 40 actcctaagc cgcctgtcgt gcatggccgc tgtagcagca cggtccaagg acccagtcct 4200 tgtggccatc atgctggctg acaccggtct tgagattctg gacagcacgt tcgtcgtgaa 4260 gaagatctcc gactcgctct ccagtctctt tcacgtgccg gcccccgt ct tcagtttcgg 4320 agctccgatt ctgttggctg ggttggtcaa agtcgcctcg agtttcttcc ggtccacacc 4380 cgaagacctt gagagagcag agaaacagct caaagcacgt gacatcaacg acatattcgc 4440 cattcttaaa aacggcgagt ggctggtcaa gctgattctt gccatccgcg actggataaa 4500 ggcatggatc gcctcagaag aaaagtttgt taccatgaca gacttggtgc ctggcatcct 4560 tgaaaagca g cgggacctca acgacccgag caagtacaaa gaagccaagg agtggctcga 4620 caacgcgcgc caagcgtgtc tgaagagcgg gaacgtccac attgccaacc tgtgcaaagt 4680 ggttgccccg gcacccagca agtcgagacc cgaacctgtg gtcgtttgcc tacg tggtaa 4740 atccggccag ggaaagagtt tccttgcgaa cgtgctcgca caagcaatct ccacccactt 4800 cactggcaga actgattcag tttggtactg cccgcctgac cctgaccact tcgacggtta 4860 caaccagcaa accgttgttg tgatggacga tttgggccag aaccccgatg gcaaggactt 4920 taaatacttt gcccagatgg tttcaaccac ggggttcatc ccgcccatgg cctcgctaga 4980 ggacaaaggc a aacctttca acagcaaggt catcattgcc accaccaatc tgtactcggg 5040 attcaccccg agaactatgg tgtgccctga tgcactgaac cggaggtttc actttgacat 5100 cgacgtgagc gccaaggacg agtacaaagt taacaacaaa ttggacataa ccaaagccct 5160 tgaagacacc cacactaacc ctgtggcaat gtttcagtac gactgtgccc ttctcaacgg 5220 catggccgtt gaaatgaaga gaatgcaaca agacgtattc aagcctttgc cacccctcca 5280 gaacgtgtac caactcgtac aggaggtgat tgaccgggtc gagcttcacg agaaagtgtc 5340 gagccggccg atctttaagc agatctcaat tccttcccaa aagtccgttt tgtacttcct 5400 cattgagaaa ggc caacacg aagcagcaat tgaattcttt gaggggatgg tgcacgactc 5460 cattaaggag gagctccggc ccctcatcca acagacgtca tttgtgaaac gcgcttttaa 5520 gcgcctgaag gaaaactttg agattgttgc cctgtgtttg actctcctgg caaacatagt 5580 gatcat gatc cgcgagactc gcaagagaca gcaaatggta gatgatgcag tgaatgagta 5640 cattgaaaag gcaaacatca ccacggatga caagaccctt gacgaggcgg aaaagaaccc 5700 tctggaaacc agtggtgcca gcaccgttgg cttcagagag agaaccctcc cgggacacaa 5760 ggcgagtgaa gacgtgaact ccgagcccgc taagaccgtg gaggaacaac cacaggctga 5820 aggaccctac gccgggccac tcgagcgtca gaaacccctg aaagtgagag ctaagctgcc 5880 acaacatgag ggaccttacg ctggcccgat ggagagacag aaaccactga aagtgaaagc 5940 aaaagccccg gtcgttaagg aaggacctta cgagggaccg gtgaagaagc ctgtcgcttt 6000 gaaagtgaaa gctaagaact tg attgtcac tgagagtggt gccccaccga ccgacttgca 6060 aaagatggtc atgagcaaca ctaagcctgt tgagctcatc cttgacggta agacggtggc 6120 catctgctgc gccaccggag tgtttggtac tgcctacctc gtgcctcgtc accttttcgc 6180 agaaaagtac gacaggatca tgttggacgg cagggccatg acagacagtg actacagagt 6240 gtttgagttt gagattaaag taaaaggaca gga catgctc tcagacgctg cgctcatggt 6300 gctccaccgt ggcaaccgtg tgagagacat cacgaaacac tttcgtgata cagcaagaat 6360 gaagaaaggt acccccgttg tcggcgtgat caacaacgcc gacgttggga gattgatttt 6420 ctccggtgag gccctcacct acaaggacat tgtagtgtgc atggatggag acaccatgcc 6480 gggcctattt gcctacagag ccgctaccaa ggctggctac tgtggaggag ccgttcttgc 6540 caaggacgga gctgacacat ttatcgtcgg cactcactcc gcaggaggca atggagtcgg 6600 gtactgctca tgcgtatcta ggtccatgct cttgaagatg aaggcacaca ttgaccccga 6660 accacaccac gaggggttga tcgttgacac cagagatgtg gaagagcgc g tgcacgtcat 6720 gcgcaaaacc aagcttgcac ccaccgtggc acacggtgtg ttcaaccctg cattcggccc 6780 cgctgccttg tccaaacaagg acccgcggct gagcgaaggg gttgtccttg atgaagtcat 6840 cttctccaaa cacaaaggag acaccaagat gtct aaggag gacaaagcgc tgttccgtcg 6900 ttgcgccgcc gactacgcgt cgcgcctgca cggcgtactg ggtacggcaa atgccccatt 6960 gagcatctac gaggcaatca agggcgttga cggactcgac gccatggaac cagacactgc 7020 gcctggtctc ccttgggccc tccaggggaa gcgccgtggc gcactgatcg actttgagaa 7080 cggcacggtc ggacccgagg ttgaggctgc cttgaagctt atggagaaaa gagaatacaa 7140 gtttgtttgt cagaccttcc tgaaagacga gattcgtccg atggagaaag tacgtgccgg 7200 caaaacccgc attgtcgacg ttttgcctgt tgaacacatt ctttacacca ggatgatgat 7260 tggcagattc tgtgctcaaa tgcactcaaa caacggaccg caaattggct cggcggtcgg 7320 ttgcaaccct gatgttgatt ggcaaagatt cggcacacac ttttcccagt acagaaacgt 7380 gtgggatgtg gactattcgg cctttgatgc taaccactgc agtgacgcaa tgaacatcat 7440 gtttgaggag gtgtttcgca cggagtttgg tttccaccca aatgctgagt ggatcctgaa 7500 gactcttgtg aacacggaac acgcctatga aaacaaaagc atcactgttg agggcggga t 7560 gccatctggt tgttccgcga caagcattat caacacaatt ttgaacaaca tttacgtgct 7620 ctacgcgttg cgtagacact atgagggagt tgagctggac acttacacca tgatctccta 7680 cggagacgac atcgtggttg caagtgatta cgatctggac ttcgaggccc tcaagcc tca 7740 cttcaaatct cttggtcaaa ccatcacccc agctgacaaa agcgacaaag gttttgttct 7800 tggtcactcc atcaccgacg tcactttcct caaaagacat ttccacatgg actatggaac 7860 tgggttttac aaacctgtga tggcctcgaa gaccctcgag gccatcctct cctttgcacg 7920 ccgtgggacc atacaggaga agttgatctc cgtggcagga ctcgccgtcc actctggacc 7980 tgacgag tac cggcgtctct ttgaaccctt tcagggtctc tttgagattc caagctacag 8040 atcactttac ctgcgttggg tgaacgccgt gtgcggtgac gcataatccc tcagatgtcg 8100 caattggcag aaagacgctg aggcgagcga cgccgtagga gtgaaaagcc caaaagggct 8 160 tttcccgctt ccttaattcc caaaaaaaaa aaaaaa 8196 <210> 2 < 211> 2199 <212> DNA <213> Artificial Sequence <220> <223> P1 of IND115 (217)/2012 (Genbank No. KT153359.1) <400> 2 ggagccgggc aatccagccc ggcgactggg tcacagaacc aatcaggcaa cactggaagc 60 atcatcaaca actactacat gcaacagtat caaaactcca tggacacaca acttggtgac 120 aatgctatca gtggaggttc caatgaaggt tccaccgaca ccac ctccac acacacaaac 180 aacacccaga ataacgattg gttttcacgc ctggctagtt ccgccttcag cggactgttt 240 ggcgcgctcc tggctgacaa gaaaacagaa gagacaactt tgcttgagga ccgcattctc 300 accaccagga atggccacac gacgtcgacg acacagt cga gcgttggcgt gacatacggt 360 tacgctgtgg ccgaggacgc ggtttcaggg cctaacactt caggcctgga gacccgtgtt 420 caacaagcgg aacggttctt caaaaagcac ttgtttgatt ggacacccaa tttggcattt 480 ggacactgct actacctgga acttcccact gaacacaaag gcgtgtacgg cagcct catg 540 gcttcgtacg cgtacatgag gaacgggtgg gacatagagg tgactgctgt tggaaaccag 600 ttcaacggcg gctgcctcct tgtcgcgctt gtgccagaat tgaaggaact tgacacacgg 660 cagaagtacc aattgactct ctttccccac cagttcatca acccgcgcac caa catgacg 720 gcccacatca acgtgccctt tgtaggtgtt aacaggtatg atcagtacga gcttcacaag 780 ccgtggacgc tcgttgtgat ggtagtggca ccactcaccg tcaagactgg tggttctgaa 840 caaattaagg tttacatgaa tgcagcacca acctatgtgc acgtggcagg ggagcttccc 900 tcgaaagagg ggatagtacc cgttgcctgc gcggacggtt atggcaacat ggtgaccacc 960 gaccc gaaaa ctgctgaccc agtctacggg aaagtgttca accccccccg gacaaatctt 1020 cccgggcgct tcacaaactt ccttgatgtt gcggaggcat gtcccacctt cctccgcttc 1080 ggagaagtac catttgtgaa gacagtgaat tctggtgacc gtttgctggc caagt ttgat 1140 gtctcgcttg ctgcggggca catgtccaac acctacttgg ccggcttggc acagtactac 1200 acgcagtaca gtggtaccat gaacattcac ttcatgttca ccgggcccac ggacgccaaa 1260 gctcggtaca tggttgccta catccctccc ggcatgacac cgcccacgga ccctgagcgt 1320 gctgcacact gcattcactc tgagtgggat actggcctta actccaagtt taccttctcc 1380 ataccctacc tctct gctgc tgactacgca tacaccgcct ctgacacggc agagactaca 1440 agtgtgcaag gttgggtgtg catctatcag attacccacg gtaaggctga aggagacgca 1500 ctggtcgttt ctgtcagcgc cggcaaagac tttgagttcc gcctgcccgt tgacgcg cgc 1560 cggcaaacca ccactaccgg cgagtcagcg gacccggtga caaccacggt tgagaactac 1620 ggaggagaaa ctcagacagc cagacggctt cacactgacg ttgccttcgt tcttgacagg 1680 ttcgtgaaac tcactacacc caagaacatt cagactcttg acctcatgca aatccctgca 1740 cacacactgg tcggagcgct gcttcggtct gcgacgtact acttctcaga cttggaggtt 1800 gcgctcgtcc acacggg ccc ggccacctgg gtgcccaacg gctcgcccaa agatgctctg 1860 gacaaccaga ctaacccaac tgcctatcag aagcaaccca ttacccgcct ggcactcccc 1920 tacaccgctc cccaccgtgt gctggcaaca gtgtacaacg ggaagacaac gtacggggaa 1980 acaacctcac ggcgtggtga catggcagcc cttgcacaaa gggtgagcag gcaactgccc 2040 acttccttta attacggcgc tgtgaaggcc gaaaacatca ctgagctttt gatccgcatg 2100 aaacgcgcgg agacatactg ccctaggcct ttgctggctc ttgacaccac tcaagaccgc 2160 cgtaagcagg agatcattgc acctgagaag cagatgatg 2199 <210> 3 <211> 633 <212> DNA <213> Artificial Sequ ence <220> <223> VP1 of BD_SI_2_2013 VP1 (Genbank KR869774.1) <400 > 3 accaccacta ccggtgagtc agcggacccg gtgacaacca cggttgagaa ctacggagga 60 gaaactcaga cagccagacg gcttcacact gacgttgcct tcgttcttga taggtttgtg 120 aaactcacta cacccaagaa cattcagact cttgacctca tgcaaatccc tgcaca caca 180 ctggtcggag cgctgcttcg gtctgcgacg tactacttct cagacttgga ggttgcgctc 240 gtccacacgg gccccgccac ctgggtgccc aacggctcgc ccaaagatgc tctggacaac 300 cagactaacc caactgccta tcagaagcaa cccattaccc gcctggcact cccctacacc 360 gctccccacc gtgtgctggc aacagtgtac aacgggaaga caacgtacgg ggaaacaacc 420 tcacggcgtg gtgacatggc agcccttgca caaagggtga gcaggcaact gcccacttcc 480 tttaattacg gcgctgtgaa ggccgaaaac atcactgagc ttttgatccg catgaaacgc 540 gcggagacat actgccctag gcctttgctg gctcttgaca ccactcaaga ccgccgta ag 600 caggagatca ttgcacctga gaagcagatg atg 633 <210> 4 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> VP1 of IND115 (217)/2012 (Genbank No. KT153359.1) 140 to 160 amino acid <400> 4 acctcacggc gtggtgacat ggcagccctt gcacaaaggg tgagcaggca actgcccact 60 tcc 63 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> VP1 of IND1 15 (217 )/2012 (Genbank No. KT153359.1) 140 to 160 amino acid sequence <400> 5 Thr Ser Arg Arg Gly Asp Met Ala Ala Leu Ala Gln Arg Val Ser Arg 1 5 10 15 Gln Leu Pro Thr Ser 20 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 6 aggaacaacc acaggctgaa ggaccttacg agggaccggt 40 <210> 7 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 7 tcggtcggtg gggcaccact ctcagtgaca atcaagttct 40 <210> 8 <211> 216 <212> DNA <213> Artificial Sequence <220> <223> Substituted 3B <400> 8 ggaccttacg agggaccggt gaagaagcct gtcgcttt ga aagtgaaagc taagaacttg 60 attgtcactg aggggccata tgaaggacca gtgaagaaac ctgtcgcttt gaaagtgaaa 120 gcaaaagccc cgattgtcac tgaaggaccc tacgagggac cggtgaagaa gcctgtcgct 180 ttgaaagtga aagccaagaa cttgattgtc actgag 216 <210> 9 <211> 72 <2 12> PRT <213> Artificial Sequence <220> <223> Substituted 3B amino acids <400> 9 Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys 1 5 10 15 Ala Lys Asn Leu Ile Val Thr Glu Gly Pro Tyr Glu Gly Pro Val Lys 20 25 30 Lys Pro Val Ala Leu Lys Val Lys Ala Lys Ala Pro Ile Val Thr Glu 35 40 45 Gly Pro Tyr Glu Gly Pro Val Lys Lys Pro Val Ala Leu Lys Val Lys 50 55 60 Ala Lys Asn Leu Ile Val Thr Glu 65 70 <210> 10 <211> 21 <212> DNA <213 > Artificial Sequence <220> <223> Primer <400> 10 accatggatg gagacaccat g 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 11 cactacaatg tccttgtagg tg 22 <210> 12 <211> 8196 <212> DNA <213> Artificial Sequence <220> <223> Asia1 Shamir virus sequence <400> 12 ttgaaagggg gcgctagggt ctcaccccta gcacgccaac gacagctcct gcattgcact 60 ccacacttac gtctgtgcac atgcgggaac cgctgg acta tcgttcaccc acctacggct 120 ggactcacgg caccgcgtgg ccatgctagc tggattgtgc ggacgaacgc catttgcgca 180 tctcgcgtgg ctggttaata ctcttaccac tttccgccta cctggtcgtt agcgctgtct 240 tgggcactcc cgttgggggc cgttcgatgc tccacggtct cccccgtgtg acaaactacg 300 gtgttgggac cgctttgtgc gagccg ctcg cctggtgtgc ttcggctgtc actcgaagcc 360 cgcctttcac cccccccccc ctaagtttta ccgtcatccc gacgttaaag ggaaccaacc 420 acaagcttgg aaccgcctcg cccgacgtga aaggggctgt aaccacaagc ttgcaccgcc 480 tttcccggcg tttaac ggga tgtaaccaca agataaacct tcgcccggaa gtaaaacgac 540 aacttcacac acttttgccc gtttttatga gaaatgggac gtctgcgcac gaaacgcgcc 600 gtcgcttgag gaatgcctgt acaaacacga ctcacgcagg tctccacaac tgacacaaac 660 cgtgcaactt gaaactccgc ctggtctttc caggtctaga ggggtgacac tttgtactgt 720 gcttgactcc acgctcggtc cactagcgag tgttagtaac agcactgttg cttcgtagcg 780 gagcatggtg gccgtgggaa cccctccttg gcgacaagga cccacggggc cgaaagccac 840 gtccttgtgg acccaccatg tgtgcaaccc cagcacggca gctctactgt gaaaacca ct 900 ttaaggtgac actgatactg gtactcagac actggtgaca ggctaaggat gcccttcagg 960 taccccgagg taacacgcga cactcgggat ctgagaaggg gactgagact tctttaaaag 1020 tgcccagttt aaaaagcttc tatgcctgaa taggcgaccg gaggccggcg cctttctcct 1 080 taactaccac ttcttaatga acacaactga ctgttttatc gctctgctgc acgccctcag 1140 agagatcaaa acgctgtttc tgtcacggac ccaagggaaa atggaattca cactctacaa 1200 cggtgagaag aagacctttt actccaggcc caacaaccac gacaactgtt ggttgaacac 1260 catcctccag ctgtttaggt acgttgatga gcctttcttt gactgggttt atgactcgcc 132 0 cgagaacctc acccttgctg caattgaaca gttggaagag gttaccggtc ttgagctgca 1380 cgaaggcggg ccacccgctc tcgtcatttg gaacatcaaa cacttgctcc acactggaat 1440 cggtactgct tcgcgaccca gcgaggtgtg tatggtggac ggcacggaca tg tgtttggc 1500 tgacttccac gccggcattt tcctgaaagg tcaggaacat gccgtgtttg cctgtgtcac 1560 ctccaacggg tggtatgcga ttgatgatga ggacttttac ccttggacgc cggacccgtc 1620 cgacgttctg gtgtttgttc cgtacgatca ggaaccgctc aacggggaat ggaaagcaaa 1680 ggttcagaaa cgacttaagg gagccggtca atccagtccg gcaaccgggt cacagaacca 1740 atctgg caac actggaagca tcattaacaa ctactacatg caacagtacc agaattccat 1800 ggacacacag cttggtgaca acgctattag cggaggttcc aacgaaggtt ccacggatac 1860 cacttccaca cacacaaaca acacccaaaa caacgactgg ttctcgcgcc tggctagctc 1920 tgcattcagt ggtctctttg gtgcactttt ggctgacaag aagacagaag agacaactct 1980 gcttgaagac cgcattctca ccaccaggaa cggccacaca acatcgacga cacagtcgag 2040 cgttggcgta acatacggtt acgctgtggc cgaggacgcg gtgtctggac ccaatacctc 2100 gggtctagag actcgtgttc aacaggcaga acggtttttc aagaaacacc tgtttgactg 2160 gacaccgaac ttggcatttg gacactgtta ctacctggaa cttcccactg aacacaagg 2220 cgtgtacggc agtctcatgg gctcgtacgc ctacatgaga aatggatggg acatagaggt 2280 gactgctgtt ggaaaccaat tcaacggtgg ttgtctcctt gtcgcgctcg tgccagagct 2340 gaaggaactc gacacgcgac agaagtacca gctgaccctc tttccccacc agttcatcaa 2400 cccacgcacc aacatgacgg cccacatcaa cgtgccgtac gtgggtatca acaggtacga 2460 ccagtacgcc ctccacaagc cgtggacgct tgttgtgatg gtggtagccc cactcaccgt 2520 caaaactggt ggttctgaac agatcaaggt ttacatgaat gcagcgccaa cctacgtgca 2580 tgtggcggga gagctgccct cgaaaga ggg aatagttccc gtcgcgtgtg cggacggtta 2640 cggcaacatg gtgactacgg acccgaagac ggccgatcca gtttacggga aagtgttcaa 2700 cccccccagg acaaacctcc ctgggcgctt cacgaacttc cttgatgttg cggaggcatg 2760 tccaacttt c ctccgctttg gagaagtacc atttgtgaag acggtgaact ctggtgaccg 2820 cttgctggcc aagttcgacg tgtccctcgc tgcagggcac atgtccaaca cctacttggc 2880 tggcctggcg cagtactaca cacagtacag cggcaccatg aacgtccact tcatgttcac 2940 cgggcccacg gatgctaaag cccgatacat ggtggcttat gtcccccctg gcatgacacc 3000 gcccacggac cctgagcacg ccgcacactg cattcactct gagtgg gata ctggtcttaa 3060 ctctaagttt accttttcca taccttacct ctctgctgct gactatgcct acactgcttc 3120 tgacgtggcg gagaccacga gtgtgcaggg atgggtgtgt atctatcaga tcacccacgg 3180 caaggctgag ggagacgcac tggtcgttt c tgtcagcgcc ggcaaagact ttgagtttcg 3240 cttgcctgtt gacgcacgcc agcaaaccac caccactggc gaatcagcag atccagtcac 3300 aaccacggtt gagaactatg gaggagagac tcagacagcc agacggcttc acactgacgt 3360 cgccttcatt cttgacaggt ttgtgaaact cactgctccc aagaacatcc aaaccctcga 3420 tctcatgcag atcccctcac acacgctggt tggagcacta cttcgttctg c gacgtacta 3480 cttctcagac ctggaggtcg cgcttgtcca cacaggcccg gtcacctggg tgcccaacgg 3540 cgcgcccaag gatgctctaa acaaccagac caacccaact gcctaccaga agcgacccat 3600 cacccgcctg gcactcccct acaccgcccc ccatcgtgtg ctggcaacag tgtacaacgg 3660 gaagacggcg tacggggaaa cgacctcaag gcgcggcgac atggcggccc tcgcacaaag 3720 gttgagcgct cggctgccca cctccttcaa ctacggcgcc gtgaaggcca acaccatcac 3780 tgagcttttg atccgcatga agcgcgcgga gacatattgc cctaggcctt tactagccct 3840 tgacaccact caggaccgcc gcaaacagga gatcattgca cctgagaagc aggtt ttgaa 3900 ctttgaccta ctcaagttgg caggagacgt tgagtccaac cctgggccct tcttcttctc 3960 cgacgttagg tcgaacttct ccaaactggt cgagaccatc aaccagatgc aggaggacat 4020 gtcaacaaag cacggacccg acttcaaccg gttggtttcc gcgtt tgagg aattggccac 4080 aggagtaaag gccatcagga acggtctcga tgaggccaag ccctggtaca agctcatcaa 4140 actcctaagc cgcctgtcgt gcatggccgc tgtagcagca cggtccaagg acccagtcct 4200 tgtggccatc atgctggctg acaccggtct tgagattctg gacagcacgt tcgtcgtgaa 4260 gaagatctcc gactcgctct ccagtctctt tcacgtgccg gcccccgtct tcagtt tcgg 4320 agctccgatt ctgttggctg ggttggtcaa agtcgcctcg agtttcttcc ggtccacacc 4380 cgaagacctt gagagagcag agaaacagct caaagcacgt gacatcaacg acatattcgc 4440 cattcttaaa aacggcgagt ggctggtcaa gctgattctt gccatccgcg actggataaa 4500 ggcatggatc gcctcagaag aaaagtttgt taccatgaca gacttggtgc ctggcatcct 4560 tgaaaagcag cgggacctca acgacccgag caagtacaaa gaagccaagg agtggctcga 4620 caacgcgcgc caagcgtgtc tgaagagcgg gaacgtccac attgccaacc tgtgcaaagt 4680 ggttgccccg gcacccagca agtcgagacc cgaacctgtg gtcgtttgcc tacgtggtaa 4 740 atccggccag ggaaagagtt tccttgcgaa cgtgctcgca caagcaatct ccacccactt 4800 cactggcaga actgattcag tttggtactg cccgcctgac cctgaccact tcgacggtta 4860 caaccagcaa accgttgttg tgatggacga tttgggccag aaccccgatg gcaagg actt 4920 taaatacttt gcccagatgg tttcaaccac ggggttcatc ccgcccatgg cctcgctaga 4980 ggacaaaggc aaacctttca acagcaaggt catcattgcc accaccaatc tgtactcggg 5040 attcaccccg agaactatgg tgtgccctga tgcactgaac cggaggtttc actttgacat 5100 cgacgtgagc gccaaggacg agtacaaagt taacaacaaa ttggacataa ccaaagccct 5160 tgaagacacc cacactaacc ctgtggcaat gtttcagtac gactgtgccc ttctcaacgg 5220 catggccgtt gaaatgaaga gaatgcaaca agacgtattc aagcctttgc cacccctcca 5280 gaacgtgtac caactcgtac aggaggtgat tgaccgggtc gagcttcacg agaaagtgtc 5340 gagccggccg atct ttaagc agatctcaat tccttcccaa aagtccgttt tgtacttcct 5400 cattgagaaa ggccaacacg aagcagcaat tgaattcttt gaggggatgg tgcacgactc 5460 cattaaggag gagctccggc ccctcatcca acagacgtca tttgtgaaac gcgcttttaa 5520 gcgcctgaag gaaaactttg agattgttgc cctgtgtttg actctcctgg caaacatagt 5580 gatcatgatc cgcgagactc gcaagagaca gcaaatggta gatgatgcag tgaatgagta 5640 cattgaaaag gcaaacatca ccacggatga caagaccctt gacgaggcgg aaaagaaccc 5700 tctggaaacc agtggtgcca gcaccgttgg cttcagagag agaaccctcc cgggacacaa 5760 ggcgagtgaa gacgtgaact ccgagcc cgc taagaccgtg gaggaacaac cacaggctga 5820 aggaccctac gccgggccac tcgagcgtca gaaacccctg aaagtgagag ctaagctgcc 5880 acaacatgag ggaccttacg ctggcccgat ggagagacag aaaccactga aagtgaaagc 5940 aaaagccccg gtcgttaagg aaggacctta cgagggaccg gtgaagaagc ctgtcgcttt 6000 gaaagtgaaa gctaagaact tgattgtcac tgagagtggt g ccccaccga ccgacttgca 6060 aaagatggtc atgagcaaca ctaagcctgt tgagctcatc cttgacggta agacggtggc 6120 catctgctgc gccaccggag tgtttggtac tgcctacctc gtgcctcgtc accttttcgc 6180 agaaaagtac gacaggatca t gttggacgg cagggccatg acagacagtg actacagagt 6240 gtttgagttt gagattaaag taaaaggaca ggacatgctc tcagacgctg cgctcatggt 6300 gctccaccgt ggcaaccgtg tgagagacat cacgaaacac tttcgtgata cagcaagaat 6360 gaagaaaggt acccccgttg tcggcgtgat caacaacgcc gacgttggga gattgatttt 6420 ctccggtgag gccctcacct acaaggacat tgtagtgtgc atggat ggag acaccatgcc 6480 gggcctattt gcctacagag ccgctaccaa ggctggctac tgtggaggag ccgttcttgc 6540 caaggacgga gctgacacat ttatcgtcgg cactcactcc gcaggaggca atggagtcgg 6600 gtactgctca tgcgtatcta ggtcagacatgct cttga tg aaggcacaca ttgaccccga 6660 accacaccac gaggggttga tcgttgacac cagagatgtg gaagagcgcg tgcacgtcat 6720 gcgcaaaacc aagcttgcac ccaccgtggc acacggtgtg ttcaaccctg cattcggccc 6780 cgctgccttg tccaaggag acccgcggct gagcgaaggg gttgtccttg atgaagtcat 6840 cttctccaaa cacaaaggag acaccaagat gtctaaggag gacaaagcgc t gttccgtcg 6900 ttgcgccgcc gactacgcgt cgcgcctgca cggcgtactg ggtacggcaa atgccccatt 6960 gagcatctac gaggcaatca agggcgttga cggactcgac gccatggaac cagacactgc 7020 gcctggtctc ccttgggccc tccaggggaa gcgccg tggc gcactgatcg actttgagaa 7080 cggcacggtc ggacccgagg ttgaggctgc cttgaagctt atggagaaaa gagaatacaa 7140 gtttgtttgt cagaccttcc tgaaagacga gattcgtccg atggagaaag tacgtgccgg 7200 caaaacccgc attgtcgacg ttttgcctgt tgaacacatt ctttacacca ggatgatgat 7260 tggcagattc tgtgctcaaa tgcactcaaa caacggaccg caaattggct cggcggtcgg 7320 t tgcaaccct gacgttgatt ggcaaagatt cggcacacac ttttcccagt acagaaacgt 7380 gtgggatgtg gactattcgg cctttgatgc taaccactgc agtgacgcaa tgaacatcat 7440 gtttgaggag gtgtttcgca cggagtttgg tttccaccca aatgctgag t ggatcctgaa 7500 gactcttgtg aacacggaac acgcctatga aaacaaaagc atcactgttg agggcgggat 7560 gccatctggt tgttccgcga caagcattat caacacaatt ttgaacaaca tttacgtgct 7620 ctacgcgttg cgtagacact atgagggagt tgagctggac acttacacca tgatctccta 7680 cggagacgac atcgtggttg caagtgatta cgatctggac ttcgaggccc tcaagcctca 7740 cttcaaatct cttggt caaa ccatcacccc agctgacaaa agcgacaaag gttttgttct 7800 tggtcactcc atcaccgacg tcactttcct caaaagacat ttccacatgg actatggaac 7860 tgggttttac aaacctgtga tggcctcgaa gaccctcgag gccatcctct cctttgcacg 7920 ccgtgg gacc atacaggaga agttgatctc cgtggcagga ctcgccgtcc actctggacc 7980 tgacgagtac cggcgtctct ttgaaccctt tcagggtctc tttgagattc caagctacag 8040 atcactttac ctgcgttggg tgaacgccgt gtgcggtgac gcataatccc tcagatgtcg 8100 caattggcag aaagacgctg aggcgagcga cgccgtagga gtgaaaagcc caaaagggct 8160 tttcccgctt ccttaatt cc caaaaaaaaa aaaaaa 8196 <210> 13 <211> 11122 <212> DNA <213> Artificial Sequence <220> <223> Plasmid for Asia1 Shamir -R <400> 13 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 ggcgaacg tg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 g ggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa agggggcgct 660 agggtctcac ccctagcacg ccaacgacag ctcctgcatt gcactccaca cttacgtct g 720 tgcacatgcg ggaaccgctg gactatcgtt cacccaccta cggctggact cacggcaccg 780 cgtggccatg ctagctggat tgtgcggacg aacgccattt gcgcatctcg cgtggctggt 840 taatactctt accactttcc gcctacctgg tcgttagcgc tgtcttgggc actcccgttg 900 ggggccgttc gatgctccac ggtctccccc gtgtgacaaa ctacggtgtt gggaccgctt 960 tgtgcgagcc g ctcgcctgg tgtgcttcgg ctgtcactcg aagcccgcct ttcacccccc 1020 cccccctaag ttttaccgtc atcccgacgt taaagggaac caaccacaag cttggaaccg 1080 cctcgcccga cgtgaaaggg gctgtaacca caagcttgca ccgcctttcc cggcgtttaa 114 0 cgggatgtaa ccacaagata aaccttcgcc cggaagtaaa acgacaactt cacacacttt 1200 tgcccgtttt tatgagaaat gggacgtctg cgcacgaaac gcgccgtcgc ttgaggaatg 1260 cctgtacaaa cacgactcac gcaggtctcc acaactgaca caaaccgtgc aacttgaaac 1320 tccgcctggt ctttccaggt ctagaggggt gacactttgt actgtgcttg actccacgct 1380 cggtccacta gcgagtgtta gtaacag cac tgttgcttcg tagcggagca tggtggccgt 1440 gggaacccct ccttggcgac aaggacccac ggggccgaaa gccacgtcct tgtggaccca 1500 ccatgtgtgc aaccccagca cggcagctct actgtgaaaa ccactttaag gtgacactga 1560 tactggtact cagacactgg t gacaggcta aggatgccct tcaggtaccc cgaggtaaca 1620 cgcgacactc gggatctgag aaggggactg agacttcttt aaaagtgccc agtttaaaaa 1680 gcttctatgc ctgaataggc gaccggaggc cggcgccttt ctccttaact accacttctt 1740 aatgaacaca actgactgtt ttatcgctct gctgcacgcc ctcagagaga tcaaaacgct 1800 gtttctgtca cggacccaag ggaaaatgga attcacactc tacaacggtg agaagaagac 1860 cttttactcc aggcccaaca accacgacaa ctgttggttg aacaccatcc tccagctgtt 1920 taggtacgtt gatgagcctt tctttgactg ggtttatgac tcgcccgaga acctcaccct 1980 tgctgcaatt gaacagttgg aagaggttac c ggtcttgag ctgcacgaag gcgggccacc 2040 cgctctcgtc atttggaaca tcaaacactt gctccacact ggaatcggta ctgcttcgcg 2100 acccagcgag gtgtgtatgg tggacggcac ggacatgtgt ttggctgact tccacgccgg 2160 cattttcctg aaaggtcagg aacatgccgt gtttgcctgt gtcacctcca acgggtggta 2220 tgcgattgat gatgaggact tttacccttg gacgccggac cc gtccgacg ttctggtgtt 2280 tgttccgtac gatcaggaac cgctcaacgg ggaatggaaa gcaaaggttc agaaacgact 2340 taagggagcc ggtcaatcca gtccggcaac cgggtcacag aaccaatctg gcaacactgg 2400 aagcatcatt aacaactact acatgcaaca gtaccagaat t ccatggaca cacagcttgg 2460 tgacaacgct attagcggag gttccaacga aggttccacg gataccactt ccacacacac 2520 aaaacacacc caaaacaacg actggttctc gcgcctggct agctctgcat tcagtggtct 2580 ctttggtgca cttttggctg acaagaagac agaagagaca actctgcttg aagaccgcat 2640 tctcaccacc aggaacggcc acacaacatc gacgacacag tcgagcgttg gcgtaacata 2700 cggttacgct gtggccgagg acgcggtgtc tggacccaat acctcgggtc tagagactcg 2760 tgttcaacag gcagaacggt ttttcaagaa acacctgttt gactggacac cgaacttggc 2820 atttggacac tgttactacc tggaacttcc cactgaacac aaaggcgtgt a cggcagtct 2880 catgggctcg tacgcctaca tgagaaatgg atgggacata gaggtgactg ctgttggaaa 2940 ccaattcaac ggtggttgtc tccttgtcgc gctcgtgcca gagctgaagg aactcgacac 3000 gcgacagaag taccagctga ccctctttcc ccaccagttc atcaacccac gcaccaacat 3060 gacggcccac atcaacgtgc cgtacgtggg tatcaacagg tacgaccagt acgccctcca 3 120 caagccgtgg acgcttgttg tgatggtggt agccccactc accgtcaaaa ctggtggttc 3180 tgaacagatc aaggtttaca tgaatgcagc gccaacctac gtgcatgtgg cgggagagct 3240 gccctcgaaa gagggaatag ttcccgtcgc gtgtgcggac ggttac ggca acatggtgac 3300 tacggacccg aagacggccg atccagttta cgggaaagtg ttcaaccccc ccagggacaaa 3360 cctccctggg cgcttcacga acttccttga tgttgcggag gcatgtccaa ctttcctccg 3420 ctttggagaa gtaccatttg tgaagacggt gaactctggt gaccgcttgc tggccaagtt 3480 cgacgtgtcc ctcgctgcag ggcacatgtc caacacctac ttggctggcc tggcgcagta 354 0 ctacacacag tacagcggca ccatgaacgt ccacttcatg ttcaccgggc ccacggatgc 3600 taaagcccga tacatggtgg cttatgtccc ccctggcatg acaccgccca cggaccctga 3660 gcacgccgca cactgcattc actctgagtg ggatactggt cttaactcta agtttacctt 37 20 ttccatacct tacctctctg ctgctgacta tgcctacact gcttctgacg tggcggagac 3780 cacgagtgtg cagggatggg tgtgtatcta tcagatcacc cacggcaagg ctgagggaga 3840 cgcactggtc gtttctgtca gcgccggcaa agactttgag tttcgcttgc ctgttgacgc 3900 acgccagcaa accaccacca ctggcgaatc agcagatcca gtcacaacca cggttgagaa 3960 ctatggagga gag actcaga cagccagacg gcttcacact gacgtcgcct tcattcttga 4020 caggtttgtg aaactcactg ctcccaagaa catccaaacc ctcgatctca tgcagatccc 4080 ctcacacacg ctggttggag cactacttcg ttctgcgacg tactacttct cagacctgga 4140 ggt cgcgctt gtccacacag gcccggtcac ctgggtgccc aacggcgcgc ccaaggatgc 4200 tctaaacaac cagaccaacc caactgccta tcagaagcaa cccatcaccc gcctggcact 4260 cccctacacc gccccccatc gtgtgctggc aacagtgtac aacgggaaga cggcgtacgg 4320 ggaaacgacc tcaaggcgcg gcgacatggc ggccctcgca caaaggttga gcgctcggct 4380 gcccacctcc ttcaactacg gcgccgt gaa ggccgacacc atcactgagc ttttgatccg 4440 catgaagcgc gcggagacat attgccctag gcctttacta gcccttgaca ccactcagga 4500 ccgccgcaaa caggagatca ttgcacctga gaagcaggtt ttgaactttg acctactcaa 4560 gttggcagga gacgttgag t ccaaccctgg gcccttcttc ttctccgacg ttaggtcgaa 4620 cttctccaaa ctggtcgaga ccatcaacca gatgcaggag gacatgtcaa caaagcacgg 4680 acccgacttc aaccggttgg tttccgcgtt tgaggaattg gccacaggag taaaggccat 4740 caggaacggt ctcgatgagg ccaagccctg gtacaagctc atcaaactcc taagccgcct 4800 gtcgtgcatg gccgctgtag cagcacggtc caaggaccca g tccttgtgg ccatcatgct 4860 ggctgacacc ggtcttgaga ttctggacag cacgttcgtc gtgaagaaga tctccgactc 4920 gctctccagt ctctttcacg tgccggcccc cgtcttcagt ttcggagctc cgattctgtt 4980 ggctgggttg gtcaaagtcg c ctcgagttt cttccggtcc acacccgaag accttgagag 5040 agcagagaaa cagctcaaag cacgtgacat caacgacata ttcgccattc ttaaaaacgg 5100 cgagtggctg gtcaagctga ttcttgccat ccgcgactgg ataaaggcat ggatcgcctc 5160 agaagaaaag tttgttacca tgacagactt ggtgcctggc atccttgaaa agcagcggga 5220 cctcaacgac ccgagcaagt acaaagaagc caaggagtgg ctcgaca acg cgcgccaagc 5280 gtgtctgaag agcgggaacg tccacattgc caacctgtgc aaagtggttg ccccggcacc 5340 cagcaagtcg agacccgaac ctgtggtcgt ttgcctacgt ggtaaatccg gccagggaaa 5400 gagtttcctt gcgaacgtgc t cgcacaagc aatctccacc cacttcactg gcagaactga 5460 ttcagtttgg tactgcccgc ctgaccctga ccacttcgac ggttacaacc agcaaaccgt 5520 tgttgtgatg gacgatttgg gccagaaccc cgatggcaag gactttaaat actttgccca 5580 gatggtttca accacggggt tcatcccgcc catggcctcg ctagaggaca aaggcaaacc 5640 tttcaacagc aaggtcatca ttgccaccac caatctgtac tcgggattca ccccgagaac 570 0 tatggtgtgc cctgatgcac tgaaccggag gtttcacttt gacatcgacg tgagcgccaa 5760 ggacgagtac aaagttaaca acaaattgga cataaccaaa gcccttgaag acacccacac 5820 taaccctgtg gcaatgtttc agtacgactg tgcccttctc aacggcatgg cc gttgaaat 5880 gaagagaatg caacaagacg tattcaagcc tttgccaccc ctccagaacg tgtaccaact 5940 cgtacaggag gtgattgacc gggtcgagct tcacgagaaa gtgtcgagcc ggccgatctt 6000 taagcagatc tcaattcctt cccaaaagtc cgttttgtac ttcctcattg agaaaggcca 6060 acacgaagca gcaattgaat tctttgaggg gatggtgcac gactccatta aggaggagct 612 0 ccggcccctc atccaacaga cgtcatttgt gaaacgcgct tttaagcgcc tgaaggaaaa 6180 ctttgagatt gttgccctgt gtttgactct cctggcaaac atagtgatca tgatccgcga 6240 gactcgcaag agacagcaaa tggtagatga tgcagtgaat gagtacattg aaaaggcaaa 6300 catcaccacg gatgacaaga cccttgacga ggcggaaaag aaccctctgg aaaccagtgg 6 360 tgccagcacc gttggcttca gagagagaac cctcccggga cacaaggcga gtgaagacgt 6420 gaactccgag cccgctaaga ccgtggagga acaaccacag gctgaaggac cctacgccgg 6480 gccactcgag cgtcagaaac ccctgaaagt gagagctaag ctgccacaac atgagggacc 65 40 ttacgctggc ccgatggaga gacagaaacc actgaaagtg aaagcaaaag ccccggtcgt 6600 taaggaagga ccttacgagg gaccggtgaa gaagcctgtc gctttgaaag tgaaagctaa 6660 gaacttgatt gtcactgaga gtggtgcccc accgaccgac ttgcaaaaga tggtcatgag 6720 caacactaag cctgttgagc tcatccttga cggtaagacg gtggccatct gctgcgccac 6780 cggagtgttt ggtactgcct acctcgtgcc tcgtcacctt ttcgcagaaa agtacgacag 6840 gatcatgttg gacggcaggg ccatgacaga cagtgactac agagtgtttg agtttgagat 6900 taaagtaaaa ggacaggaca tgctctcaga cgctgcgctc atggt gctcc accgtggcaa 6960 ccgtgtgaga gacatcacga aacactttcg tgatacagca agaatgaaga aaggtacccc 7020 cgttgtcggc gtgatcaaca acgccgacgt tgggagattg attttctccg gtgaggccct 7080 cacctacaag gacattgtag tgtgcatgga tggagacacc atgccgggcc tatttgccta 7140 cagagccgct accaaggctg gctactgtgg aggagccgtt cttgccaagg acggagctga 7200 cacatttatc gtcggc actc actccgcagg aggcaatgga gtcgggtact gctcatgcgt 7260 atctaggtcc atgctcttga agatgaaggc acacattgac cccgaaccac accacgaggg 7320 gttgatcgtt gacaccagag atgtggaaga gcgcgtgcac gtcatgcgca aaaccaagct 7380 tgcacccacc gtggcacacg gtgtgttcaa ccctgcattc ggccccgctg ccttgtccaa 7440 caaggacccg cggctgagcg aaggggttgt ccttgatgaa gtcatcttct ccaaacacaa 7500 aggagacacc aagatgtcta aggaggacaa agcgctgttc cgtcgttgcg ccgccgacta 7560 cgcgtcgcgc ctgcacggcg tactgggtac ggcaaatgcc ccattgagca tctacgaggc 7620 aatca agggc gttgacggac tcgacgccat ggaaccagac actgcgcctg gtctcccttg 7680 ggccctccag gggaagcgcc gtggcgcact gatcgacttt gagaacggca cggtcggacc 7740 cgaggttgag gctgccttga agcttatgga gaaaagagaa tacaagtttg tttgtca gac 7800 cttcctgaaa gacgagattc gtccgatgga gaaagtacgt gccggcaaaa cccgcattgt 7860 cgacgttttg cctgttgaac acattcttta caccaggatg atgattggca gattctgtgc 7920 tcaaatgcac tcaaacaacg gaccgcaaat tggctcggcg gtcggttgca accctgacgt 7980 tgattggcaa agattcggca cacacttttc ccagtacaga aacgtgtggg atgtggacta 8040 ttcggccttt gatgctaacc actg cagtga cgcaatgaac atcatgtttg aggaggtgtt 8100 tcgcacggag tttggtttcc acccaaatgc tgagtggatc ctgaagactc ttgtgaacac 8160 ggaacacgcc tatgaaaaca aaagcatcac tgttgagggc gggatgccat ctggttgttc 8220 cg cgacaagc attatcaaca caattttgaa caacatttac gtgctctacg cgttgcgtag 8280 acactatgag ggagttgagc tggacactta caccatgatc tcctacggag acgacatcgt 8340 ggttgcaagt gattacgatc tggacttcga ggccctcaag cctcacttca aatctcttgg 8400 tcaaaccatc accccagctg acaaaagcga caaaggtttt gttcttggtc actccatcac 8460 cgacgtcact ttcctcaaaa gacatttcca catggactat gga actgggt tttacaaacc 8520 tgtgatggcc tcgaagaccc tcgaggccat cctctccttt gcacgccgtg ggaccataca 8580 ggagaagttg atctccgtgg caggactcgc cgtccactct ggacctgacg agtaccggcg 8640 tctctttgaa ccctttcagg gtctctttga gatt ccaagc tacagatcac tttacctgcg 8700 ttgggtgaac gccgtgtgcg gtgacgcata atccctcaga tgtcgcaatt ggcagaaaga 8760 cgctgaggcg agcgacgccg taggagtgaa aagcccaaaa gggcttttcc cgcttcctta 8820 attcccaaaa aaaaaaaaaa actagttcta gagcggccgc caccgcggtg gagctccagc 8880 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatgg tc atagctgttt 8940 cctgtgtgaa attgttatcc gctcacaatt ccacaaaca tacgagccgg aagcataaag 9000 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 9060 cccgctttcc agtcgggaaa cctgtcgtgc cagct gcatt aatgaatcgg ccaacgcgcg 9120 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 9180 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 9240 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 9300 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc t gacgagcat 9360 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 9420 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 9480 tacctgtccg cctttctccc ttcgggaagc gtggcgctt t ctcatagctc acgctgtagg 9540 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 9600 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 9660 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 9720 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 978 0 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaaa gagttggtag ctcttgatcc 9840 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 9900 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cg ctcagtgg 9960 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 10020 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 10080 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 10140 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 10200 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 10260 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 10320 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 10380 ttg cgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 10440 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 10500 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 10560 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 10620 tgcttttctg tg actggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 10680 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 10740 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 10800 tt gagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 10860 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 10920 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 10980 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaaacaa 11040 ataggggttc cgcgcacatt tccccgaaaa gtg ccacata aacaaatagg ggttccgcgc 11100 acatttcccc gaaaagtgcc ac 11122 <210> 14 <211> 8268 <212> DNA <213> Artificial Sequence <220> <223 > As Sham G VIII EP I <400> 14 ttgaaagggg gcgctagggt ctcaccccta gcacgccaac gacagctcct gcattgcact 60 ccacacttac gtctgtgcac atgcgggaac cgctggacta tcgttcaccc acctacggct 120 ggactcacgg caccgcgtgg ccatgctagc tggattg tgc ggacgaacgc catttgcgca 180 tctcgcgtgg ctggttaata ctcttaccac tttccgccta cctggtcgtt agcgctgtct 240 tgggcactcc cgttgggggc cgttcgatgc tccacggtct cccccgtgtg acaaactacg 300 gtgttgggac cgct ttgtgc gagccgctcg cctggtgtgc ttcggctgtc actcgaagcc 360 cgcctttcac cccccccccc cctaagtttt accgtcatcc cgacgttaaa gggaaccaac 420 cacaagcttg gaaccgcctc gcccgacgtg aaaggggctg taaccacaag cttgcaccgc 480 ctttcccggc gtttaacggg atgtaaccac a agataaacc ttcgcccgga agtaaaacga 540 caacttcaca cacttttgcc cgtttttatg agaaatggga cgtctgcgca cgaaacgcgc 600 cgtcgcttga ggaatgcctg tacaaacacg actcacgcag gtctccacaa ctgacacaaa 660 ccgtgcaact tgaaactccg c ctggtcttt ccaggtctag aggggtgaca ctttgtactg 720 tgcttgactc cacgctcggt ccactagcga gtgttagtaa cagcactgtt gcttcgtagc 780 ggagcatggt ggccgtggga acccctcctt ggcgacaagg acccacgggg ccgaaagcca 840 cgtccttgtg gacccaccat gtgtgcaacc ccagcacggc agctctactg tgaaaaccac 900 tttaaggtga cactgatact ggtactcaga cactggtgac agg ctaagga tgcccttcag 960 gtaccccgag gtaacacgcg acactcggga tctgagaagg ggactgagac ttctttaaaa 1020 gtgcccagtt taaaaagctt ctatgcctga ataggcgacc ggaggccggc gcctttctcc 1080 ttaactacca cttcttaatg aacacaactg actgttttat cgctctgctg cacgccctca 1140 gagagatcaa aacgctgttt ctgtcacgga cccaagggaa aatggaattc acactctaca 1200 acggtgagaa gaagaccttt tactccaggc ccaacaacca cgacaactgt tggttgaaca 1260 ccatcctcca gctgtttagg tacgttgatg agcctttctt tgactgggtt tatgactcgc 1320 ccgagaacct cacccttgct gcaattgaac agttggaaga ggttaccgg t cttgagctgc 1380 acgaaggcgg gccacccgct ctcgtcattt ggaacatcaa acacttgctc cacactggaa 1440 tcggtactgc ttcgcgaccc agcgaggtgt gtatggtgga cggcacggac atgtgtttgg 1500 ctgacttcca cgccggcatt ttcctga aag gtcaggaaca tgccgtgttt gcctgtgtca 1560 cctccaacgg gtggtatgcg attgatgatg aggactttta cccttggacg ccggacccgt 1620 ccgacgttct ggtgtttgtt ccgtacgatc aggaaccgct caacggggaa tggaaagcaa 1680 aggttcagaa acgacttaag ggagccggtc aatccagtcc ggcaaccggg tcacagaacc 1740 aatctggcaa cactggaagc atcattaaca actactacat gcaacagtac cagaattcca 1800 tggacacaca gcttggtgac aacgctatta gcggaggttc caacgaaggt tccacggata 1860 ccacttccac acacacaaac aacacccaaa acaacgactg gttctcgcgc ctggctagct 1920 ctgcattcag tggtctcttt ggtgcacttt tggctgacaa gaagacagaa gagacaactc 1980 tgcttgaaga ccgcattctc accaccagga acggccacac aacatcgacg acacagtcga 2040 gcgttggcgt aacatacggt tacgctgtgg ccgaggacgc ggtgtctgga cccaatacct 2100 cgggtctaga gactcgtgtt caacaggcag aacggttttt caagaaacac ctgtttgact 2160 ggacaccgaa cttggcattt ggacactgtt actacctgga acttcccact gaacacaaag 22 20 gcgtgtacgg cagtctcatg ggctcgtacg cctacatgag aaatggatgg gacatagagg 2280 tgactgctgt tggaaaccaa ttcaacggtg gttgtctcct tgtcgcgctc gtgccagagc 2340 tgaaggaact cgacacgcga cagaagtacc agctgaccct ctttccccac ca gttcatca 2400 acccacgcac caacatgacg gcccacatca acgtgccgta cgtgggtatc aacaggtacg 2460 accagtacgc cctccacaag ccgtggacgc ttgttgtgat ggtggtagcc ccactcaccg 2520 tcaaaactgg tggttctgaa cagatcaagg tttacatgaa tgcagcgcca acctacgtgc 2580 atgtggcggg agagctgccc tcgaaagagg gaatagttcc cgtcgcgtgt gcggacggtt 2 640 acggcaacat ggtgactacg gacccgaaga cggccgatcc agtttacggg aaagtgttca 2700 acccccccag gacaaacctc cctgggcgct tcacgaactt ccttgatgtt gcggaggcat 2760 gtccaacttt cctccgcttt ggagaagtac catttgtgaa gacggtgaac tctggtgacc 2820 gcttgctggc caagttcgac gtgtccctcg ctgcagggca catgtccaac acctacttgg 2880 ctggcctggc gcagtactac acacagtaca gcggcaccat gaacgtccac ttcatgttca 2940 ccgggcccac ggatgctaaa gcccgataca tggtggctta tgtcccccct ggcatgacac 3000 cgcccacgga ccctgagcac gccgcacact gcattcactc tgagtgggat actggtctta 3060 actctaagtt tacctttt cc ataccttacc tctctgctgc tgactatgcc tacactgctt 3120 ctgacgtggc ggagaccacg agtgtgcagg gatgggtgtg tatctatcag atcacccacg 3180 gcaaggctga gggagacgca ctggtcgttt ctgtcagcgc cggcaaagac tttgagtttc 32 40 gcttgcctgt tgacgcacgc cagcaaacca ccaccactgg cgaatcagca gatccagtca 3300 caaccacggt tgagaactat ggaggagaga ctcagacagc cagacggctt cacactgacg 3360 tcgccttcat tcttgacagg tttgtgaaac tcactgctcc caagaacatc caaaccctcg 3420 atctcatgca gatcccctca cacacgctgg ttggagcact acttcgttct gcgacgtact 3480 acttctcaga cctggaggtc gcgcttgtcc aca caggccc ggtcacctgg gtgcccaacg 3540 gcgcgcccaa ggatgctcta aacaaccaga ccaacccaac tgcctatcag aagcaaccca 3600 tcacccgcct ggcactcccc tacaccgccc cccatcgtgt gctggcaaca gtgtacaacg 3660 ggaagacggc gtacggggaa a cgacctcaa ggcgcggcga catggcggcc ctcgcaacct 3720 cacggcgtgg tgacatggca gcccttgcac aaagggtgag caggcaactg cccacttccc 3780 aaaggttgag cgctcggctg cccacctcct tcaactacgg cgccgtgaag gccgacacca 3840 tcactgagct tttgatccgc atgaagcgcg cggagacata ttgccctagg cctttactag 3900 cccttgacac cactcaggac cgccgcaaac aggagatcat tgcacct gag aagcaggttt 3960 tgaactttga cctactcaag ttggcaggag acgttgagtc caaccctggg cccttcttct 4020 tctccgacgt taggtcgaac ttctccaaac tggtcgagac catcaaccag atgcaggagg 4080 acatgtcaac aaagcacgga cccgacttca accggttggt ttccgcgttt gaggaattgg 4140 ccacaggagt aaaggccatc aggaacggtc tcgatgaggc caagccctgg tacaagctca 4200 tcaaactcct aagccgcctg tcgtgcatgg ccgctgtagc agcacggtcc aaggacccag 4260 tccttgtggc catcatgctg gctgacaccg gtcttgagat tctggacagc acgttcgtcg 4320 tgaagaagat ctccgactcg ctctccagtc tctttcacgt gccggccccc gtcttcagtt 4380 tcggagctcc gattctgttg gctgggttgg tcaaagtcgc ctcgagtttc ttccggtcca 4440 cacccgaaga ccttgagaga gcagagaaac agctcaaagc acgtgacatc aacgacatat 4500 tcgccattct taaaaacggc gagtggct gg tcaagctgat tcttgccatc cgcgactgga 4560 taaaggcatg gatcgcctca gaagaaaagt ttgttaccat gacagacttg gtgcctggca 4620 tccttgaaaa gcagcgggac ctcaacgacc cgagcaagta caaagaagcc aaggagtggc 4680 tcgacaacgc gcgccaagcg tgtctgaaga gcgggaacgt ccacattgcc aacctgtgca 4740 aagtggttgc cccggcaccc agcaagtcga gacccgaacc tgtggtcgt t tgcctacgtg 4800 gtaaatccgg ccagggaaag agtttccttg cgaacgtgct cgcacaagca atctccaccc 4860 acttcactgg cagaactgat tcagtttggt actgcccgcc tgaccctgac cacttcgacg 4920 gttacaacca gcaaaccgtt gttgtgatgg acgatttgg g ccagaaccc gatggcaagg 4980 actttaaata ctttgcccag atggtttcaa ccacggggtt catcccgccc atggcctcgc 5040 tagaggacaa aggcaaacct ttcaacagca aggtcatcat tgccaccacc aatctgtact 5100 cgggattcac cccgagaact atggtgtgcc ctgatgcact gaaccggagg tttcactttg 5160 acatcgacgt gagcgccaag gacgagtaca aagttaacaa caaattggac ataaccaaag 5220 cccttgaaga c acccacact aaccctgtgg caatgtttca gtacgactgt gcccttctca 5280 acggcatggc cgttgaaatg aagagaatgc aacaagacgt attcaagcct ttgccacccc 5340 tccagaacgt gtaccaactc gtacaggagg tgattgaccg ggtcgagctt cacgagaaag 5400 tg tcgagccg gccgatcttt aagcagatct caattccttc ccaaaagtcc gttttgtact 5460 tcctcattga gaaaggccaa cacgaagcag caattgaatt ctttgagggg atggtgcacg 5520 actccattaa ggaggagctc cggcccctca tccaacagac gtcatttgtg aaacgcgctt 5580 ttaagcgcct gaaggaaaac tttgagattg ttgccctgtg tttgactctc ctggcaaaca 5640 tagtgatcat g atccgcgag actcgcaaga gacagcaaat ggtagatgat gcagtgaatg 5700 agtacattga aaaggcaaac atcaccacgg atgacaagac ccttgacgag gcggaaaaga 5760 accctctgga aaccagtggt gccagcaccg ttggcttcag agagagaacc ctcccgggac 5820 acaaggcgag tgaagacgtg aactccgagc ccgctaagac cgtggaggaa caaccacagg 5880 ctgaaggacc ttacgaggga ccggtgaaga agcctgtcgc tttgaaagtg aaagctaaga 5940 acttgattgt cactgagggg ccatatgaag gaccagtgaa gaaacctgtc gctttgaaag 6000 tgaaagcaaa agccccgatt gtcactgaag gaccctacga gggaccggtg aagaagcctg 6060 tcgctttgaa agtga aagcc aagaacttga ttgtcactga gagtggtgcc ccaccgaccg 6120 acttgcaaaa gatggtcatg agcaacacta agcctgttga gctcatcctt gacggtaaga 6180 cggtggccat ctgctgcgcc accggagtgt ttggtactgc ctacctcgtg cctcgtcacc 6240 ttt tcgcaga aaagtacgac aggatcatgt tggacggcag ggccatgaca gacagtgact 6300 acagagtgtt tgagtttgag attaaagtaa aaggacagga catgctctca gacgctgcgc 6360 tcatggtgct ccaccgtggc aaccgtgtga gagacatcac gaaacacttt cgtgatacag 6420 caagaatgaa gaaaggtacc cccgttgtcg gcgtgatcaa caacgccgac gttgggagat 6480 tgattttctc cggtgaggcc ct cacctaca aggacattgt agtgaccatg gatggagaca 6540 ccatgccggg cctatttgcc tacagagccg ctaccaaggc tggctactgt ggaggagccg 6600 ttcttgccaa ggacggagct gacacattta tcgtcggcac tcactccgca ggaggcaatg 6660 gagtcgggta ctgctcatgc g tatctaggt ccatgctctt gaagatgaag gcacacattg 6720 accccgaacc acaccacgag gggttgatcg ttgacaccag agatgtggaa gagcgcgtgc 6780 acgtcatgcg caaaaccaag cttgcaccca ccgtggcaca cggtgtgttc aaccctgcat 6840 tcggccccgc tgccttgtcc aacaaggacc cgcggctgag cgaaggggtt gtccttgatg 6900 aagtcatctt ctccaaacac aaaggagaca ccaagatgtc taaggaggac aaagcgctgt 6960 tccgtcgttg cgccgccgac tacgcgtcgc gcctgcacgg cgtactgggt acggcaaatg 7020 ccccattgag catctacgag gcaatcaagg gcgttgacgg actcgacgcc atggaaccag 7080 acactgcgcc t ggtctccct tgggccctcc aggggaagcg ccgtggcgca ctgatcgact 7140 ttgagaacgg cacggtcgga cccgaggttg aggctgcctt gaagcttatg gagaaaagag 7200 aatacaagtt tgtttgtcag accttcctga aagacgagat tcgtccgatg gagaaagtac 7260 gtgccggcaa aacccgcatt gtcgacgttt tgcctgttga acacattctt tacaccagga 7320 tgatgattgg cagattctgt gctcaaatgc actcaaacaa cggac cgcaa attggctcgg 7380 cggtcggttg caaccctgac gttgattggc aaagattcgg cacacacttt tcccagtaca 7440 gaaacgtgtg ggatgtggac tattcggcct ttgatgctaa ccactgcagt gacgcaatga 7500 acatcatgtt tgaggaggtg tttcgc acgg agtttggttt ccacccaaat gctgagtgga 7560 tcctgaagac tcttgtgaac acggaacacg cctatgaaaa caaaagcatc actgttgagg 7620 gcgggatgcc atctggttgt tccgcgacaa gcattatcaa cacaattttg aacaacattt 7680 acgtgctcta cgcgttgcgt agacactatg agggagttga gctggacact tacaccatga 7740 tctcctacgg agacgacatc gtggttgcaa gtgattacga tctggacttc gagg ccctca 7800 agcctcactt caaatctctt ggtcaaacca tcaccccagc tgacaaaagc gacaaaggtt 7860 ttgttcttgg tcactccatc accgacgtca ctttcctcaa aagacatttc cacatggact 7920 atggaactgg gttttacaaa cctgtgatgg cctcgaagac cctcgaggcc atcctctcct 7980 ttgcacgccg tgggaccata caggagaagt tgatctccgt ggcaggactc gccgtccact 8040 ctggacctga cgagtaccgg cgtctctttg aaccctttca gggtctcttt gagattccaa 8100 gctacagatc actttacctg cgttgggtga acgccgtgtg cggtgacgca taatccctca 8160 gatgtcgcaa ttggcagaaa gacgctgagg cgagcgacgc cgtaggagtg aaaagcccaa 8220 aagggctttt cccgcttcct taattcccaa aaaaaaaaaaa aaaaaaaaa 8268 <210> 15 <211> 8210 <212> DNA <213> Artificial Sequence <220> <223> As Sham G VIII VP1 Virus <400> 15 ttgaaagggg gcgctagggt ctcaccccta gcacgccaac gacagctcct gcattgcact 60 ccacacttac gtctgtgcac atgcgggaac cgctggacta tcgttcaccc acctacggct 120 ggactcacgg caccgcgtgg ccatgctagc tggatt gtgc ggacgaacgc catttgcgca 180 tctcgcgtgg ctggttaata ctcttaccac tttccgccta cctggtcgtt agcgctgtct 240 tgggcactcc cgttgggggc cgttcgatgc tccacggtct cccccgtgtg acaaactacg 300 gtgttgggac cg ctttgtgc gagccgctcg cctggtgtgc ttcggctgtc actcgaagcc 360 cgcctttcac cccccccccc ctaagtttta ccgtcatccc gacgttaaag ggaaccaacc 420 acaagcttgg aaccgcctcg cccgacgtga aaggggctgt aaccacaagc ttgcaccgcc 480 tttcccggcg tttaacggga tgtaaccaca agataaacct tcgcccggaa gtaaaacgac 540 aacttcacac acttttgccc gtttttatga gaaatgggac gtctgcgcac gaaacgcgcc 600 gtcgcttgag gaatgcctgt acaaacacga ctcacgcagg tctccacaac tgacacaaac 660 cgtgcaactt gaaactccgc ctggtctttc caggtctaga gg gg tgacac tttgtactgt 720 gcttgactcc acgctcggtc cactagcgag tgttagtaac agcactgttg cttcgtagcg 780 gagcatggtg gccgtgggaa cccctccttg gcgacaagga cccacggggc cgaaagccac 840 gtccttgtgg acccaccatg tgtgcaaccc cagcacggca gctctactgt gaaaaccact 900 ttaaggtgac actgatactg gtactcagac actggtgaca ggctaaggat gcccttcagg 960 taccccgagg taac acgcga cactcgggat ctgagaaggg gactgagact tctttaaaag 1020 tgcccagttt aaaaagcttc tatgcctgaa taggcgaccg gaggccggcg cctttctcct 1080 taactaccac ttcttaatga acacaactga ctgttttatc gctctgctgc acgccctcag 1140 agagatcaaa acgctgtttc tgtcacggac ccaagggaaa atggaattca cactctacaa 1200 cggtgagaag aagacctttt actccaggcc caacaaccac gacaactgtt ggttgaacac 1260 catcctccag ctgtttaggt acgttgatga gcctttcttt gactgggttt atgactcgcc 1320 cgagaacctc acccttgctg caattgaaca gttggaagag gttaccggtc ttgagctgca 1380 cgaaggcg gg ccacccgctc tcgtcatttg gaacatcaaa cacttgctcc acactggaat 1440 cggtactgct tcgcgaccca gcgaggtgtg tatggtggac ggcacggaca tgtgtttggc 1500 tgacttccac gccggcattt tcctgaaagg tcaggaacat gccgtgtttg cctgtgt cac 1560 ctccaacggg tggtatgcga ttgatgatga ggacttttac ccttggacgc cggacccgtc 1620 cgacgttctg gtgtttgttc cgtacgatca ggaaccgctc aacggggaat ggaaagcaaa 1680 ggttcagaaa cgacttaagg gagccggtca atccagtccg gcaaccgggt cacagaacca 1740 atctggcaac actggaagca tcattaacaa ctactacatg caacagtacc agaattccat 1800 ggacacacag cttggtgaca acgctattag cggaggt tcc aacgaaggtt ccacggatac 1860 cacttccaca cacacaaaca acacccaaaa caacgactgg ttctcgcgcc tggctagctc 1920 tgcattcagt ggtctctttg gtgcactttt ggctgacaag aagacagaag agacaactct 1980 gcttgaagac cgcattctca ccaccaggaa cggcc acaca acatcgacga cacagtcgag 2040 cgttggcgta acatacggtt acgctgtggc cgaggacgcg gtgtctggac ccaatacctc 2100 gggtctagag actcgtgttc aacaggcaga acggtttttc aagaaacacc tgtttgactg 2160 gacaccgaac ttggcatttg gacactgtta ctacctggaa cttcccactg aacacaagg 2220 cgtgtacggc agtctcatgg gctc gtacgc ctacatgaga aatggatggg acatagaggt 2280 gactgctgtt ggaaaccaat tcaacggtgg ttgtctcctt gtcgcgctcg tgccagagct 2340 gaaggaactc gacacgcgac agaagtacca gctgaccctc tttccccacc agttcatcaa 2400 cccacgcacc aacatg acgg cccacatcaa cgtgccgtac gtgggtatca acaggtacga 2460 ccagtacgcc ctccacaagc cgtggacgct tgttgtgatg gtggtagccc cactcaccgt 2520 caaaactggt ggttctgaac agatcaaggt ttacatgaat gcagcgccaa cctacgtgca 2580 tgtggcggga gagctgccct cgaaagaggg aatagttccc gtcgcgtgtg cggacggtta 2640 cggcaacatg gtgactacgg acccgaagac ggccgatcca gtttacggga aagtgttcaa 2700 cccccccagg acaaacctcc ctgggcgctt cacgaacttc cttgatgttg cggaggcatg 2760 tccaactttc ctccgctttg gagaagtacc atttgtgaag acggtgaact ctggtgaccg 2820 cttgctggcc aagttcgacg tgtccctcgc tgcagggcac atgtccaaca cctacttggc 2880 tggcctggcg cagtactaca cacagtacag cggcaccatg aacgtccact tcatgttcac 2940 cgggcccacg gatgctaaag cccgatacat ggtggcttat gtcccccctg gcatgacacc 3000 gcccacggac cctgagcacg ccgcacactg cattcactct gagtgggata ctggtcttaa 3060 ctctaagttt accttttcca taccttacct ctctgctgct gactatgcct a cactgcttc 3120 tgacgtggcg gagaccacga gtgtgcaggg atgggtgtgt atctatcaga tcacccacgg 3180 caaggctgag ggagacgcac tggtcgtttc tgtcagcgcc ggcaaagact ttgagtttcg 3240 cttgcctgtt gacgcacgcc agcaa accac cactaccggt gagtcagcgg acccggtgac 3300 aaccacggtt gagaactacg gaggagaaac tcagacagcc agacggcttc acactgacgt 3360 tgccttcgtt cttgataggt ttgtgaaact cactacaccc aagaacattc agactcttga 3420 cctcatgcaa atccctgcac acacactggt cggagcgctg cttcggtctg cgacgtacta 3480 cttctcagac ttggaggttg cgctcgtcca cacgggcccc gccacctggg tgcccaacgg 3540 ctcgcccaaa gatgctctgg acaaccagac taacccaact gcctatcaga agcaacccat 3600 tacccgcctg gcactcccct acaccgctcc ccaccgtgtg ctggcaacag tgtacaacgg 3660 gaagacaacg tacggggaaa caacctcacg gcgtggtgac atggcag ccc ttgcacaaag 3720 ggtgagcagg caactgccca cttcctttaa ttacggcgct gtgaaggccg aaaaacatcac 3780 tgagcttttg atccgcatga aacgcgcgga gacatactgc cctaggcctt tgctggctct 3840 tgacaccact caagaccgcc gtaagcagga gatcattgca cctgagaagc agatgatggt 3900 tttgaacttt gacctactca agttggcagg agacgttgag tccaaccctg ggcccttctt 3960 ct tctccgac gttaggtcga acttctccaa actggtcgag accatcaacc agatgcagga 4020 ggacatgtca acaaagcacg gacccgactt caaccggttg gtttccgcgt ttgaggaatt 4080 ggccacagga gtaaaggcca tcaggaacgg tctcgatgag gccaagccct ggtacaagct 4 140 catcaaactc ctaagccgcc tgtcgtgcat ggccgctgta gcagcacggt ccaaggaccc 4200 agtccttgtg gccatcatgc tggctgacac cggtcttgag attctggaca gcacgttcgt 4260 cgtgaagaag atctccgact cgctctccag tctctttcac gtgccggccc ccgtcttcag 4320 tttcggagct ccgattctgt tggctgggtt ggtcaaagtc gcctcgagtt tcttccggtc 43 80 cacacccgaa gaccttgaga gagcagagaa acagctcaaa gcacgtgaca tcaacgacat 4440 attcgccatt cttaaaaacg gcgagtggct ggtcaagctg attcttgcca tccgcgactg 4500 gataaaggca tggatcgcct cagaagaaaa gtttgttacc atgacagact tggtgcctgg 4 560 catccttgaa aagcagcggg acctcaacga cccgagcaag tacaaagaag ccaaggagtg 4620 gctcgacaac gcgcgccaag cgtgtctgaa gagcgggaac gtccacattg ccaacctgtg 4680 caaagtggtt gccccggcac ccagcaagtc gagacccgaa cctgtggtcg tttgcctacg 4740 tggtaaatcc ggccagggaa agagtttcct tgcgaacgtg ctcgcacaag caatctccac 4800 ccacttcact ggcaga actg attcagtttg gtactgcccg cctgaccctg accacttcga 4860 cggttacaac cagcaaaccg ttgttgtgat ggacgatttg ggccagaacc ccgatggcaa 4920 ggactttaaa tactttgccc agatggtttc aaccacgggg ttcatcccgc ccatggcctc 4980 gctaga ggac aaaggcaaac ctttcaacag caaggtcatc attgccacca ccaatctgta 5040 ctcgggattc accccgagaa ctatggtgtg ccctgatgca ctgaaccgga ggtttcactt 5100 tgacatcgac gtgagcgcca aggacgagta caaagttaac aacaaattgg acataaccaa 5160 agcccttgaa gacacccaca ctaaccctgt ggcaatgttt cagtacgact gtgcccttct 5220 caacggcatg gccgttgaaa tgaagagaat gca acaagac gtattcaagc ctttgccacc 5280 cctccagaac gtgtaccaac tcgtacagga ggtgattgac cgggtcgagc ttcacgagaa 5340 agtgtcgagc cggccgatct ttaagcagat ctcaattcct tcccaaaagt ccgttttgta 5400 cttcctcatt gagaaa ggcc aacacgaagc agcaattgaa ttctttgagg ggatggtgca 5460 cgactccatt aaggaggagc tccggcccct catccaacag acgtcatttg tgaaacgcgc 5520 ttttaagcgc ctgaaggaaa actttgagat tgttgccctg tgtttgactc tcctggcaaa 5580 catagtgatc atgatccgcg agactcgcaa gagacagcaa atggtagatg atgcagtgaa 5640 tgagtacatt gaaaaggcaa acatcaccac ggatgaca ag acccttgacg aggcggaaaa 5700 gaaccctctg gaaaccagtg gtgccagcac cgttggcttc agagagagaa ccctcccggg 5760 acacaaggcg agtgaagacg tgaactccga gcccgctaag accgtggagg aacaaccaca 5820 ggctgaagga ccttacgagg gaccgg tgaa gaagcctgtc gctttgaaag tgaaagctaa 5880 gaacttgatt gtcactgagg ggccatatga aggaccagtg aagaaacctg tcgctttgaa 5940 agtgaaagca aaagccccga ttgtcactga aggaccctac gagggaccgg tgaagaagcc 6000 tgtcgctttg aaagtgaaag ccaagaactt gattgtcact gagagtggtg ccccaccgac 6060 cgacttgcaa aagatggtca tgagcaacac taagcctgtt gagctcatcc ttgac ggtaa 6120 gacggtggcc atctgctgcg ccaccggagt gtttggtact gcctacctcg tgcctcgtca 6180 ccttttcgca gaaaagtacg acaggatcat gttggacggc agggccatga cagacagtga 6240 ctacagagtg tttgagtttg agattaaagt aaaaggac ag gacatgctct cagacgctgc 6300 gctcatggtg ctccaccgtg gcaaccgtgt gagagacatc acgaaacact ttcgtgatac 6360 agcaagaatg aagaaaggta cccccgttgt cggcgtgatc aacaacgccg acgttgggag 6420 attgattttc tccggtgagg ccctcaccta caaggacatt gtagtgacca tggatggaga 6480 caccatgccg ggcctatttg cctacagagc cgctaccaag gctggctact gtggaggagc 6 540 cgttcttgcc aaggacggag ctgacacatt tatcgtcggc actcactccg caggaggcaa 6600 tggagtcggg tactgctcat gcgtatctag gtccatgctc ttgaagatga aggcacacat 6660 tgaccccgaa ccacaccacg aggggttgat cgttgacacc agagatgtgg aagag cgcgt 6720 gcacgtcatg cgcaaaacca agcttgcacc caccgtggca cacggtgtgt tcaaccctgc 6780 attcggcccc gctgccttgt ccaacaagga cccgcggctg agcgaagggg ttgtccttga 6840 tgaagtcatc ttctccaaac acaaaggaga caccaagatg tctaaggagg acaaagcgct 6900 gttccgtcgt tgcgccgccg actacgcgtc gcgcctgcac ggcgtactgg gtacgg caaa 6960 tgccccattg agcatctacg aggcaatcaa gggcgttgac ggactcgacg ccatggaacc 7020 agacactgcg cctggtctcc cttgggccct ccagggggaag cgccgtggcg cactgatcga 7080 ctttgagaac ggcacggtcg gacccgaggt tgaggctgcc ttga agctta tggagaaaag 7140 agaatacaag tttgtttgtc agaccttcct gaaagacgag attcgtccga tggagaaagt 7200 acgtgccggc aaaacccgca ttgtcgacgt tttgcctgtt gaacacattc tttacaccag 7260 gatgatgatt ggcagattct gtgctcaaat gcactcaaac aacggaccgc aaattggctc 7320 ggcggtcggt tgcaaccctg acgttgattg gcaaagattc ggcacacact tttcccagta 7380 cagaaacgtg tgggatgtgg actattcggc ctttgatgct aaccactgca gtgacgcaat 7440 gaacatcatg tttgaggagg tgtttcgcac ggagtttggt ttccacccaa atgctgagtg 7500 gatcctgaag actcttgtga acacggaaca cgcctatgaa aacaaaagca tc actgttga 7560 gggcgggatg ccatctggtt gttccgcgac aagcattatc aacacaattt tgaacaacat 7620 ttacgtgctc tacgcgttgc gtagacacta tgagggagtt gagctggaca cttacaccat 7680 gatctcctac ggagacgaca tcgtggttgc aagtgattac gatctggact tcgaggccct 7740 caagcctcac ttcaaatctc ttggtcaaac catcacccca gctgacaaaa gcgacaaagg 7800 ttttgttctt ggtcactcca t caccgacgt cactttcctc aaaagacatt tccacatgga 7860 ctatggaact gggttttaca aacctgtgat ggcctcgaag accctcgagg ccatcctctc 7920 ctttgcacgc cgtgggacca tacaggagaa gttgatctcc gtggcaggac tcgccgtcca 7980 ctctggacct g acgagtacc ggcgtctctt tgaacccttt cagggtctct ttgagattcc 8040 aagctacaga tcactttacc tgcgttgggt gaacgccgtg tgcggtgacg cataatccct 8100 cagatgtcgc aattggcaga aagacgctga ggcgagcgac gccgtaggag tgaaaagccc 8160aaaagggctt ttcccgcttc cttaattccc aaaaaaaaaaa aaaaaaaaaa 8210

Claims (5)

Asia1 type foot-and-mouth disease recombinant virus consisting of the sequence of SEQ ID NO. 12. A recombinant plasmid consisting of the sequence of SEQ ID NO: 13 used to produce the Asia1 type foot-and-mouth disease recombinant virus of claim 1. Foot-and-mouth disease 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 virus antigen of claim 3. A method of preventing, improving or treating foot-and-mouth disease using the vaccine composition of claim 4.