KR102269272B1 - Recombinant viruses expressing the foot-and-mouth disease type A protective antigen which inserted long term immunity enhancing gene, and a vaccine composition comprising the same - Google Patents

Abstract

The present invention relates to a foot-and-mouth disease recombinant virus expressing a foot-and-mouth disease type A protective antigen and a vaccine composition comprising the same, and a recombinant foot-and-mouth disease virus and long-term high titer that can be usefully used as a vaccine strain having strong protection against foot-and-mouth disease type A virus It is possible to provide a foot-and-mouth disease vaccine composition capable of inducing protective antibodies.

Description

Recombinant viruses expressing the foot-and-mouth disease type A protective antigen which inserted long term immunity enhancing gene, and a vaccine composition comprising the same}

The present invention relates to a recombinant virus expressing a foot-and-mouth disease type A protective antigen and a vaccine composition comprising the same, specifically, a recombinant virus expressing a foot-and-mouth disease type A protective antigen into which an HSP70 gene, which is a gene for enhancing long-term immunity, is inserted, and a vaccine composition comprising the same is about

Foot-and-mouth disease (FMD) is a highly contagious viral disease that mainly affects cloven hoofed livestock, and causes serious economic loss to the livestock industry due to its rapid spread, high mortality and reduced productivity. Susceptible species consist of more than 70 wildlife species, including ruminants (livestock ruminants) such as cattle, pigs, buffalo, camels, sheep, and goats. The disease is accompanied by high fever and is known to cause blistering in the mouth, tongue, snout, nose, nipples, hooves and other hairless areas of the skin. According to the Livestock Infectious Diseases Prevention Act, it is a first-class livestock infectious disease, classified and designated as a management target by the World Organization for Animal Health (OIE), and must be reported to the OIE when it occurs.

Foot-and-mouth disease pathogen (foot and mouth disease virus) is a single-stranded bipolar RNA virus belonging to the genus Picornaviridae and Aphthovirus and has seven different serotypes (A, O, C, Asia1, SAT 1, SAT 2, SAT 3). When the nucleotide sequence similarity of the gene regions corresponding to the VP1 protein of the foot-and-mouth disease virus to each other is about 85% or more, one classification is formed. Viruses of different serotypes are not serologically neutralized and have large genetic or antigenic differences so that cross-protection is not achieved by vaccines.

Therefore, in order to protect against each of these serotypes and regional types, all vaccine strains must be developed and used, and for this purpose, the outdoor strains must be continuously cultured, adapted to cells and maintained at a titer above a certain level. However, this method is time consuming, and objective data on the efficacy of vaccines including these vaccine strains are unclear. Therefore, it is necessary to prepare a recombinant virus using a vaccine strain that has a lot of previously reported information and proven in terms of protective efficacy, so that the necessary vaccine strain can be rapidly developed and used at an appropriate time.

In addition, the foot-and-mouth disease vaccine has disadvantages such as short persistence of antibody and low immunogenicity in pigs after inoculation, and the protective ability by humoral immune response is not perfect. Therefore, in order to overcome the limitations of the existing foot-and-mouth disease vaccine, innovative solutions such as increasing the antigen's own immunogenicity, long-term induction of protective antibodies, and simultaneous induction of cellular and humoral immune responses are needed by the immune-enhancing foot-and-mouth disease vaccine strain.

In order to solve the above problems, the present inventors have developed an HSP70 gene capable of inducing long-term lasting immunity and memory response using a basic vector composed of viral genes based on weakened foot-and-mouth disease virus type A, which is already known as a foot-and-mouth disease vaccine strain. An immune-enhancing recombinant virus of a new strategy was manufactured, and a foot-and-mouth disease vaccine composition capable of inducing long-term high-titer protective antibody containing inactivated antigen isolated and purified therefrom as an active ingredient was developed.

An object of the present invention is to provide a recombinant foot-and-mouth disease virus that can be usefully used as a vaccine strain having strong protection against the foot-and-mouth disease type A virus by inserting the HSP70 gene that induces long-term immunity and memory response into the foot-and-mouth disease type A virus. have.

In addition, it is to provide a method for preparing the recombinant virus, isolation, and purification of the antigen, and to provide a vaccine composition for preventing foot-and-mouth disease comprising the same as an active ingredient.

In order to achieve the above object, in the present invention, after replacing P1 of O1 Manisa with P1 of A22/Iraq/24/64, VP1 amino acid residue (residue) SEQ ID NO: 152-171 (RGD 6th amino acid) An object of the present invention is to provide a foot-and-mouth disease recombinant virus into which the HSP70 gene is inserted and an antigen for foot-and-mouth disease virus obtained by purifying and isolating the recombinant foot-and-mouth disease virus.

In addition, the present invention is to provide a recombinant plasmid containing the recombinant foot-and-mouth disease virus.

Another object of the present invention is to provide a vaccine composition comprising the foot-and-mouth disease recombinant virus or a foot-and-mouth disease virus antigen obtained by purifying and isolating the recombinant foot-and-mouth disease virus.

Another object of the present invention is to provide a foot-and-mouth disease diagnostic kit comprising the foot-and-mouth disease recombinant virus or the foot-and-mouth disease virus antigen.

Another object of the present invention is to provide a method for diagnosing foot-and-mouth disease using the foot-and-mouth disease diagnostic kit.

Another object of the present invention is to provide a method for preventing and treating foot-and-mouth disease by using the recombinant foot-and-mouth disease virus or a vaccine composition comprising the recombinant foot-and-mouth disease virus antigen.

As used herein, the term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. A vector can be a plasmid, a phage particle or simply a potential genomic insert. Upon transformation into an appropriate host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Since a plasmid is currently the most commonly used form of vector, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention.

For the purposes of the present invention, it is preferred to use a plasmid vector. Typical plasmid vectors that can be used for this purpose include (a) an initiation point of replication that allows efficient replication to include hundreds of plasmid vectors per host cell, and (b) a selection of host cells transformed with the plasmid vector. It has a structure including a selection label 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 foreign DNA can be easily ligated by using a synthetic oligonucleotide adapter or linker according to a conventional method.

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

The cells of the present invention are canines, felines, boars, boars, deer, giraffidae, peccaryids, camelids, hippocampi, equine, tapir, rhinoceros, weasel, lepidoptera, It may be derived from one or more 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), pig At least one selected from the group consisting of kidney cells (IBRS-2) and bovine kidney cells (LFBK) may be used.

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

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

The foot-and-mouth disease vaccine composition of the present invention includes 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.

The foot-and-mouth disease recombinant virus or antigen isolated and purified from the recombinant virus may be administered at a dose of 1/640 to 1/10, preferably at a dose of 1/40 to 1/10.

The present invention relates to a foot-and-mouth disease recombinant virus expressing a foot-and-mouth disease type A protective antigen and a vaccine composition comprising the same, and a recombinant foot-and-mouth disease virus and long-term high titer that can be usefully used as a vaccine strain having strong protection against foot-and-mouth disease type A virus It is possible to provide a foot-and-mouth disease vaccine composition capable of inducing protective antibodies.

1 shows a schematic diagram of the genome of a recombinant foot-and-mouth disease virus in which the protective antigen of foot-and-mouth disease A serotype is expressed.
Figure 2 shows a schematic diagram of a recombinant plasmid in which the HSP70 gene nucleotide sequence is inserted into the foot-and-mouth disease type A vaccine strain A22/Iraq/24/64.
Figure 3 shows the result of confirming the virus particles (146S) confirmed through an electron microscope (TEM) image of the recombinant virus newly prepared from the plasmid of Figure 2 .
4 shows the protective effect (survival rate and body weight change) of mice by inoculation with a test vaccine containing A-HSP70 antigen and challenge inoculation with FMDV (A Malay 97).
5 shows the results of SP A ELISA antibody levels in pig serum for antibodies immunized with serum obtained through an immune test in pigs.
6 shows the antibody titers neutralizing foot-and-mouth disease virus in bovine serum against antibodies immunized with serum obtained through an immunization test in pigs.
7 shows the expression of the antigen in the confirmed A-HSP70-R foot-and-mouth disease virus-infected cells with a simple antigen kit (PBM kit, USA; SP (structural protein) band is formed by band formation, NSP (non-structural protein, non-structural protein) -Structural protein (not formed) shows the results.

Hereinafter, the present invention is described in detail by 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.

<Example 1> Recombinant plasmid construction

The whole foot-and-mouth disease O-Manisa virus gene (GenBank Accession No. AY593823.1) was amplified by PCR, and the O-Manisa gene was inserted into the plasmid (pBluescript SK II) to construct a recombinant plasmid pO-Manisa (SEQ ID NO: 1) .

Among the recombinant plasmids (pO-Manisa) prepared above, the gene encoding the protective antigen P1 protein (sequences 2356 to 4557 of SEQ ID NO: 1) was transferred to the foot-and-mouth disease A serotype virus A22/Iraq/24/64 (GenBank Accession No. AY593764.1), a plasmid into which the recombinant gene was inserted (pOm-A22-P1) was prepared by substituting a gene (SEQ ID NO: 2) encoding the protective antigen P1 protein (SEQ ID NO: 3).

Based on the plasmid (pOm-A22-P1) prepared above, the amino acid residue SEQ ID NO: 152-171 (RGD 6th amino acid) of VP1, the HSP70 sequence between the 4380 and 4381 bases of SEQ ID NO: 3 (CAACCGTCGGTGCAGATCCAGGTCTATCAGGGGGAGCGTGAGATCGCCGCGCACAACAAG ( SEQ ID NO: 4) was inserted, and the method is as follows; (The amino acid corresponding to SEQ ID NO: 4 is QPSVQIQVYQGEREIAAHNK (SEQ ID NO: 5))

The above plasmid (300ng/μl), primer HSP70 F (5'-GGGGAGCGTGAGATCGCCGCGCACAACAAGGCGAGGGTCGCCGCTCAGCT-3' (SEQ ID NO: 6), 10 pmol/μl, 1 μl) and primer HSP70 R (5'-CTGATAGACCTGGATCTGCACCGACGGTTGCGTCGCAGAGole SEQ ID NO: 7GGTTCGTCCGAGAGGC) /μl, 1ul) was used. Conditions for PCR were 5x phusion HF buffer (Thermo scientific, Waltham, MA, USA. Cat. No: 00709661, 10 μl), 10 mM dNTP (Invitrogen, Carlsbad, CA, USA. Cat. No: 1722464, 1 μl), phusion DNA Polymerase (2U/μl, Thermo scientific, Waltham, MA, USA. Cat. No: 00607531 μl), after 98°C 30 seconds with a volume of sterile distilled water (35μl), 98°C 20 seconds, 65°C 20 seconds, 72°C 2 The reaction was carried out at 25 cycles for 30 seconds and a final 72° C. for 10 minutes. Then, 1 μl of Dpn1 (Enzynomics, Daejeon, Korea. Cat. No: R054S) was combined with the PCR product (25 μl) and reacted in an incubator at 37°C for 1 hour. Dpn1 treated product (4μl) with sterile distilled water (35μl), Ligation high (TOYOBO, Osaka, Japan. Cat. No: 75677, 5μl), Polynucelo kinase T4 (5U/μl, TOYOBO, Osaka, Japan. Cat. No : 812400, 1 μl) and ligation was performed in a water bath at 16° C. for 1 hour. After performing Ligation, Com. Cell DH5α (Yeastern Biotech, Taipei, Taiwan. Cat. No: RC901, 100 μl) was ligated with the plasmid (9 μl) on ice for 5 minutes, 42° C. 45 seconds, and 2 minutes on ice, followed by SOC (Sigma-Aldrich, St Louis, MO, USA. Cat. No: S1797, 200 μl) and incubated for 1 hour at 37° C. in a shaker incubator. After plating on an agar plate containing ampicillin, it was incubated at 37°C overnight, sterile distilled water (18 μl), forward universal primer VP1 (5'-AGNGCNGGNAARTTTGA-3' (SEQ ID NO: 8), 100 pmol/μl, 1 μl in a Colony PCR tube. ), reverse universal After mixing primer VP1 (5'-CATGTCNTCCATCTGGTT-3' (SEQ ID NO: 9), 100 pmol/μl, 1 μl) and colonies with tip tip, lightly scrape and mix at 94°C for 5 minutes, 94°C for 30 seconds, 55°C for 30 seconds, 72°C PCR was performed at 25 cycles for 1 minute, and finally at 72°C for 5 minutes. the universal In the primer, N means any base is allowed.

PCR sample (5μl) and 6x loading buffer (DYNE BIO, Gyeonggi-do, Korea. Cat. No: 50910218, 1μl) were mixed and loaded on agarose gel, while 100bp maker (DYNE BIO, Gyeonggi-do, Korea) 5 μl of Cat. No: 501904218) was loaded onto an agarose gel.

After performing electrophoresis at 100V for 30 minutes, the band was confirmed in Gel doc. ExoSAP (Thermo scientific, Waltham, MA, USA. Cat. No: 00747996, 2 μl) was mixed with the PCR product (5 μl) for which the band was confirmed, and then PCR was performed at 37° C. for 15 minutes and 85° C. for 15 minutes. The insertion of HSP70 into the amino acid sequence 152-171 (RGD 6th amino acid) in VP1 was confirmed through DNA full sequencing. The colony whose sequence was confirmed was placed in 200 ml of LB media containing ampicillin and cultured overnight in a shaker incubator at 37°C, and the plasmid of SEQ ID NO: 10 was prepared using Midi prep (MACHEREY-NAGEL, Duren, Germany. Cat. No: 740414). prepared (Fig. 2).

<Example 2> Preparation of immuno-enhancing recombinant virus

For recovery of the recombinant foot-and-mouth disease virus, the recombinant plasmid prepared in Example 1 was transduced into BHKT7-9 (cell line expressing T7 RNA polymerase) using Lipo3000 (Invitrogen, Carlsbad, CA, USA. Cat. No: L3000001). After culturing for 2 to 3 days, the formed recombinant virus was prepared (FIG. 3). Thereafter, the prepared virus was inoculated in ZZ-R (fetal goat tongue) cells or BHK-21 (juvenile hamster kidney) cells through serial passage to propagate the virus.

<Example 3> Purification of antigen using recombinant virus

Antigens purified and inactivated according to the following method were used;

It was prepared from the FMDV recombinant virus of Example 2 and BHK-21 cells. For virus infection, the culture medium was substituted with serum-free Dulbecco Modified Eagle medium (DMEM, Cellgro, Manassas, VA, USA) and _{cultured in 5% CO 2} gas at 37° C. for 1 hour to inoculate the virus. Extracellular virus was removed. 24 hours after infection, the virus was inactivated by treatment twice for 24 hours with 0.003N binary ethylenimine in a shaker incubator, followed by polyethylene glycol (PEG) 6000 (Sigma-Aldrich, St. Louis, MO, USA). concentrated. The virus concentrate was layered on a 15-45% sucrose density gradient and centrifuged. After ultracentrifugation, the bottom of the centrifuge tube was drilled and 1 mL fractions were collected. The presence of FMDV particles in each fraction sample was confirmed by optical density using a lateral flow device (BioSign FMDV Ag, Princeton BioMeditech, Princeton, NJ, USA). Before use in the experiment, the pre-PEG-treated supernatant was passed through ZZ-R and BHK-21 cells at least twice to ensure that no cytopathic effect (CPE) occurred, thereby confirming that the supernatant was free of live virus.

<Experimental Example 1> Confirmation of immunogenicity in mice

1-1. mouse

Age- and sex-matched wild-type C57BL/6 mice (7-week-old females) were purchased (KOSA BIO Inc., Gyeonggi-do, Korea). All rats were housed in micro-isolated cages in a specific sterile pathogen (SPF) animal facility at Biosafety Level 3 (ABSL3) of the Ministry of Agriculture, Food and Rural Affairs.

1-2. Vaccination and foot-and-mouth disease virus attack

In order to confirm the immunogenicity of the antigen isolated and purified from the long-term immunity-enhancing foot-and-mouth disease virus A-HSP70, to evaluate its potential as a foot-and-mouth disease vaccine strain, and to evaluate the protective effect against FMDV infection, the experiment was performed with the following strategy. The vaccine composition used in the experiment was as follows; Antigen of Example 3 (15 μg/dose/ml, 1/10 to 1/640 dose), ISA 206 (50%, w/w), 10% Al(OH) ₃ , 15 μg/mouse of Quil-A.

Mice were vaccinated by IM (intramuscular, intramuscular inoculation) at 0 dpv (days post vaccination), then 7 days (56 days post vaccination, dpv), FMDV (100LD ₅₀ of A Malay 97, SEA topotype) was administered intraperitoneally. After administration, the survival rate and weight change were monitored until 7 days post challenge (dpc), as shown in FIG. 4 .

As a result of the experiment, the test vaccine using the antigen isolated and purified from A-HSP70, a recombinant foot-and-mouth disease virus for enhancing long-term immunity, showed a survival rate of 100% at 1/10 dose, 80% at 1/40 dose, and 80% at 1/10 dose when inoculated into mice. The 1/160 and 1/640 doses showed a 0% survival rate, but compared to the negative control group, mortality was delayed. In weight loss, almost no body weight change was observed at 1/10 and 1/40 doses, so it was judged to play an important role in host defense.

<Experimental Example 2> Vaccination and sampling in pigs

In order to observe the long-term immune effect of the long-term immune-enhancing recombinant virus of the present invention in pigs, the following experiment was performed.

The vaccine composition of the positive control group was as follows; A22 recombinant antigen (15 μg/dose/ml), ISA 206 (50%, w/w), 10% prepared in the same manner as in Examples 2 and 3 except that HSP70 was not inserted in Example 1 Al(OH) ₃ , 150 μg/dose/ml Quil-A.

The vaccine composition of the experimental group was as follows; Antigen of Example 3 (15 μg/dose/ml), ISA 206 (50%, w/w), 10% Al(OH) ₃ , 150 μg/dose/ml Quil-A.

Using 10-12 week old pigs, animals without foot-and-mouth disease antibody were administered twice (0dpv, 28dpv) at intervals of 28 days, 1ml vaccine was administered through the deep intramuscular route in the neck of the animal. Blood samples were collected at 0, 14, 28, 42, 56, 70, 84 dpv of pigs.

Animals were monitored daily for body temperature and appetite. Serum samples were stored at -80 °C until testing was performed.

<Experimental Example 3> Serum test

3-1. ELISA (enzyme-linked immunosorbent assay) for detection of structural protein antibodies

For the detection of the structural protein (SP) antibody in serum, PrioCHECK FMDV type O (Prionics, Switzerland) was used. The absorbance of the ELISA plate was converted to a percentage inhibition (PI) value. When the PI value was 50% or higher, the animal was considered antibody positive, and the results are shown in FIG. 5 .

As a result of the experiment, the experimental group administered with the antigen of the present invention exhibited an antibody formation rate of 30% or more on the 14th day after vaccine administration, and maintained a steady antibody formation rate of 60% or more from day 42 to day 84. On the other hand, the positive control group showed an antibody formation rate of 20% on the 14th day after vaccine administration, and the antibody formation rate gradually increased, but the highest antibody formation rate was about 40%. Therefore, it can be seen that the vaccine according to the present invention has a high antibody formation rate as well as a long-term immune enhancing effect.

3-2. Virus Neutralization Test

Virus neutralization test was performed according to the OIE manual. It was activated by bathing in serum at 56°C for 30 minutes. Two-fold serial dilutions of serum samples from 1:4 to 1:512 were prepared by adjusting the cell density to form a 70% monolayer. The diluted serum samples were incubated with 100 tissue culture infectious dose (TCID) 50/0.5ml homolog virus at 37°C for 1 hour. After 1 hour, LF-BK (bovine kidney) cell suspension was added to all wells. After 2-3 days, the CPE was measured to determine the titer _{to neutralize 100TCID 50} of the virus as _{log 10} of the titer antibody dilution, and the results are shown in FIG. 6 .

As a result of the test, the test vaccine using the A-HSP70 antigen, a long-term immunity-enhancing recombinant foot-and-mouth disease virus, showed an increase in antibody titer and VN titer from 14 dpv when inoculated to outdoor pigs, and after boosting at 28 dpv, up to 84 dpv compared to the positive control. A significantly high VN titer was maintained ( p <0.001).

<Experimental Example 4> Differentiation from outdoor liquor using a simple kit including the present invention

A simple antigen kit (PBM kit, USA; SP (structural protein)) containing the recombinant virus of the present invention was used to test whether antigen expression in cells infected with foot-and-mouth disease virus could be differentiated from an outdoor strain. The band was confirmed by band formation and NSP (non-structural protein; non-formation) As a result of the experiment, it is possible to differentiate from the field strain in terms of antigen and serology through the non-formation of NSP band. appeared, 2.34 It was found that it was possible to differentiate even with a small amount of ng (1/640 dose) ( FIG. 7 ).

<110> Animal and Plant Quarantine Agency <120> Recombinant viruses expressing the foot-and-mouth disease type A protective antigen which inserted long term immunity enhancing gene, and a vaccine composition comprising the same <130> 19-11311 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 11097 <212> DNA <213> Artificial Sequence <220> <223> pO-Manisa plasmid <400> 1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600 taaaacgacg gccagtgagc atatgtaata cgactcacta tagggttgaa agggggcgct 660 agggtctcac ccctagcatg ccaacgacag ctcctacgtc gcactccaca ctaacgtttg 720 tgtgcgcgcg ggaaccgatg gacttttgtt cacccaccta cagttggact cacggcaccg 780 cgtggccatt ttagctgggt tgtgcggacg aacactgctt gcgcatctcg cgtgaccggt 840 tagtactctt accactatcc gcctacttgg tcgttagcgc tgtcctgggc actcttgttg 900 ggggctgttc aacgctctac ggtctcccct gcgtaacaga ctacggtgtt ggggccgctt 960 cgtgcgagcc gatcgcttgg tgtgcctcgg ctgtcgcccg aagcccgcct ttcacccccc 1020 cccccccccc ccccctaggt tttaccgtcg ttcccgacgt taatggggaa acaaccacaa 1080 gcttaacacc gtcttgcccg acgtaaaagg gctgcaacca aaaagcttgt gccgcctttc 1140 ccggcgttaa tgggaggtaa ccacaagaca aaccttcacc cggaagtaaa acggcaactt 1200 cacacagttt tgcccgtttt cgtgagaaat gggccgtcaa cgcacgaaac gcgccgtcgc 1260 ttgaggagga cttgtacaaa cacgatctat gcaggtttcc acaactgaca caaaccgtgc 1320 aacttgaaac cccgcctggt ctttccaggt ctagaggggc gacattttgt actgtgcttg 1380 actccacgct cggtccacta gcgagtgtta gtagtagcac tgttgcttcg tagcggagca 1440 tgatggccgt gggagcttcc ccttggtaac aaggacccac ggggccaaaa gccacgtcct 1500 accggaccca tcatgtgtgc aaacccagca cggcaacttt actgcgaaaa ccactttaag 1560 gtgacactga tactggtact caatcactgg tgacaggcta aggatgccct tcaggtaccc 1620 cgaggtaaca cgcgacactc gggatctgag aaggggactg gggcttcttt aaaagtgccc 1680 agtttaaaaa gcttctatgc ctgaataggc gaccggaggc cggcgccttt tcactgtttt 1740 actactgttt tcatgaatac aactgactgt ttcaccgccc tgttacacgc tctcagagag 1800 atcaaaacac tgtttctttt acggacacaa ggaaagatgg aattcacact ttacaacggt 1860 gagaagaaaa ccttctactc cagacccaac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggtatgt tgatgagcct ttctttgact gggtctacga ctcgcctgaa 1980 aacctcactc ttgaggcaat caaacagttg gaagagacaa ccggtcttga gctgcacgag 2040 ggtggaccac ccgctctcgt catctggaac atcaaacact tgcttcacac cggaatcggc 2100 actgcctcac gccctagcga ggtgtgtatg gtggacggaa cggacatgtg tttagctgat 2160 tttcatgctg gcattttcct gaaaggacag gaacatgctg tgttcgcctg tgtcacctcc 2220 aacgggtggt acgcgattga tgacgaggac ttttaccctt ggacaccgga cccgtccgac 2280 gttctggtgt ttgtcccgta cgatcaagaa ccgcttaacg gagagtggaa aacaaaggtc 2340 cagaaaaggc tcaagggagc cgggcaatcc agcccggcaa ccgggtcaca gaaccaatca 2400 ggcaacactg ggagcatcat caacaattac tacatgcagc agtaccaaaa ctccatggac 2460 acacaacttg gtgacaacgc tacaagcgga ggctcaaacg aggggtccac ggacacaacc 2520 tccacccaca caaccaacac tcagaacaac gactggttct cgaagctggc cagttccgct 2580 ttcagcggtc ttttcggcgc tcttctcgcc gacaagaaaa ccgaggagac cactcttctc 2640 gaggaccgca tcctcactac tcgtaacgga cacaccacct cgacaaccca gtcgagcgtt 2700 ggagtcacgt acgggtatgc aacagctgag gatttcgtga gcgggccaaa cacctctggt 2760 ctcgagacca gggttgccca ggcagagcgg ttctttaaaa cccacctgtt cgactgggtc 2820 accagtgacc cattcggacg gtgccacctg ctggaacttc caactgacca caaaggtgtc 2880 tacggcagcc tgaccgactc gtatgcttat atgaggaacg gctgggatgt tgaagtcact 2940 gcagtgggaa accagttcaa tggaggatgc ctgttggtgg ccatggtgcc agaactttgc 3000 tccatacaga agagggagct gtaccagctc acgctctttc ctcaccagtt catcaaccct 3060 cggacgaaca tgacagcaca catcactgtg ccctttgttg gcgtcaaccg ttatgaccag 3120 tacaaggtac acaaaccttg gaccctcgtg gttatggttg tagcccccct gactgtcaac 3180 agtgaaggtg ccccgcaaat caaggtgtat gccaacatcg cacctaccaa cgtacacgtc 3240 gcgggtgagt tcccttccaa agaggggatc ttccctgtgg cttgcagcga tggttatggc 3300 ggtctggtga ccactgaccc gaaaacggct gaccccgctt acgggaaagt gtttaacccc 3360 ccccgcaaca tgttgccggg gcggttcacc aattttcttg acgtggctga ggcgtgcccc 3420 acgtttctcc acttcgaggg tgacgtgcca tacgtgacca cgaagacgga ttcagacagg 3480 gtgctcgctc agttcgactt gtctttggca gcaaagcaca tgtcgaacac cttccttgca 3540 ggtctcgccc agtactacac acagtacagc ggcaccatca acctgcactt catgttcaca 3600 gggcctactg acgcgaaggc gcgttacatg attgcgtatg ctcctcctgg catggaacca 3660 cctaaaacgc cagaggcggc tgcccactgc attcatgctg aatgggacac agggttgaac 3720 tcaaaattca cattttcaat cccttacctt tcggcggctg attacgctta cacagcgtct 3780 gacactgctg agaccacaaa tgtacaggga tgggtttgcc tgtttcaaat aacacacggg 3840 aaagctgacg gcgacgcact ggtcgttttg gctagcgccg gaaaggactt tgagctgcgc 3900 ctgccggtgg atgctcgcac acagactacc tccgcgggcg agtcagctga ccccgtgacc 3960 gccaccgttg agaattacgg tggcgagaca caggtccaga ggcgccaaca cacggacgtc 4020 tcatttatat tagacagatt tgtgacagtg acaccaaaag accaaattaa tgtattggac 4080 ctgatgcaaa cccctgctca cactttggtg ggagcactcc ttcgtactgc cacttactat 4140 ttcgctgact tagaggtggc agtgaagcac aagggaaacc tcacctgggt cccgaacggg 4200 gcgcctgaag cggcgttgga caacaccacc aacccaacag cttaccacaa ggcaccactc 4260 acccgacttg cactgcctta cacggcgcca caccgcgtgt tggctactgt ttacaacggg 4320 aactgcaagt atggtgacgg cacggtggcc aatgtgagag gtgacctgca agtgttggcc 4380 cagaaggcgg cgagagcgct gcctacctcc ttcaactacg gtgccattaa agctactcgg 4440 gtgactgaac tgctttaccg catgaagagg gctgagacat actgcccccg gcctcttttg 4500 gccattcacc cggaccaggc tagacacaag cagaagattg tggcaccggt ggaacagctt 4560 ctaaattttg acctgctcaa attggcggga gatgtggagt ccaaccctgg gcccttcttc 4620 ttctccgacg tcaggtcaaa tttctcaaaa ctggtagaaa ccatcaatca gatgcaggag 4680 gacatgtcaa caaaacacgg gcctgacttt aaccggttgg tgtccgcatt tgaggaattg 4740 gccactggag tgaaggctat cagggccggt ctcgacgagg ccaaaccctg gtacaaactc 4800 atcaagctcc tgagccgctt gtcgtgcatg gccgctgtag cagcacggtc aaaggaccca 4860 gtccttgtgg ccatcatgct ggctgacacc ggtcttgaga ttctggacag cacctttgtc 4920 gtgaagaaga tctccgactc gctctccagt ctctttcacg tgccggcccc cgtcttcagt 4980 ttcggagccc cgattctgtt ggccgggttg gtcaaagtcg cctcgagttt cttccggtcc 5040 acacccgaag accttgagag agcagaaaaa cagctcaaag cacgtgacat taacgacata 5100 ttcgccattc tcaagaacgg cgagtggctg gtcaagctga tccttgccat ccgcgactgg 5160 atcaaagcgt ggatcgcctc agaagaaaag tttgtcacca tgacggactt ggtgcctggt 5220 atccttgaaa agcagcggga tctcaacgac ccgagtaagt acaaggaagc caaggagtgg 5280 ctcgacaacg cgcgccaggc gtgtttgaag agcgggaacg ttcacattgc caatttgtgc 5340 aaagtggtcg ccccggcacc cagcaagtcg agacccgaac ccgtggtcgt ttgcctccgc 5400 ggcaaatccg gccaggggaa gagtttcctt gcgaacgtgc tcgcgcaagc aatctccacc 5460 cacttcaccg gcagaactga ttcggtttgg tactgcccgc ctgaccctga ccacttcgac 5520 ggttacaacc agcagaccgt tgtcgtgatg gacgatttgg gccagaaccc cgatggcaag 5580 gacttcaagt acttcgccca gatggtttcg accacggggt tcatcccgcc catggcctcg 5640 cttgaggaca aaggcaagcc tttcaacagc aaagtcatca ttgctaccac caacctgtac 5700 tcgggtttca ccccgagaac aatggtgtgt cctgacgcgc tgaaccggag gttccacttt 5760 gacatcgacg tgagtgccaa ggacgggtac aaagttaaca acaaattgga cataatcaaa 5820 gctcttgaag acacccacac caacccagtg gcgatgttcc aatacgactg tgcccttcta 5880 aacggtatgg cagttgaaat gaagagaatg caacaggata tgttcaagcc tcaaccaccc 5940 ctccagaacg tgtaccaact cgttcacgag gtgattgaac gggtcgagct ccacgagaag 6000 gtgtcgagcc acccgatttt caaacagata tcaattcctt cccaaaagtc tgtgttgtac 6060 ttcctcattg agaaaggcca acacgaagca gcaattgaat tctttgaggg aatggtgcat 6120 gactccatca aggaagagct ccggcccctc atccaacaga cctcatttgt gaaacgcgct 6180 tttaagcgcc tgaaggaaaa ctttgagact gttgccctgt gtttgactct tttggcaaac 6240 atagtgatca tgatccgcga gactcgcaag agacaacaga tggtggacga tgcagtgaat 6300 gactacattg agaaggcaaa catcaccaca gatgacaaga ctcttgacga ggcggaaaag 6360 aaccctctag agaccagcgg tgccagcact attggtttca gagagagaac tctcccgggg 6420 cacaaggcga gcgatgacgt gagctccgag cccgccaaac ccgtggagga ccgaccacaa 6480 gctgaagggc cctacgccgg accacttgag cgtcagaaac ctctgagagt gaaaaccaag 6540 ttgccacaac aggagggacc ctacgctggc ccgatggata gacagaaacc gttgaaagtg 6600 agagcaagag ccccggtcgt gaaggaggga ccctacgagg gaccggtgaa gaagcctgtc 6660 gctttgaaag tgaaagccaa gaacttgatt gtcactgaga gtggtgcccc accgaccgac 6720 ttgcagaaga tggtcatggg caacactaag cctgttgagc tcatcctcga cgggaagacg 6780 gtagccatct gctgtgctac cggagtgttt ggcactgcct acctcgtacc tcgtcacctc 6840 ttcgcggaga agtacgacaa gataatgttg gacggtagag ccatgacaga cagtgactac 6900 agagtgtttg agtttgagat taaagtaaaa ggacaggaca tgctctcaga cgctgcactc 6960 atggtgcttc accgtgggaa ccgcgtgaga gacatcacga aacattttcg tgacacagca 7020 agaatgaaga aaggcacccc cgttgtcggt gtgatcaaca acgccgacgt tgggagactg 7080 attttctctg gagaggccct tacctacaaa gacatgtag tgtgcatgga tggagacacc 7140 atgccgggcc tgtttgccta cagagccgcc accaaggctg gttactgcgg gggagccgtt 7200 ctcgccaagg acggagccga cacatcatc gttggcaccc actccgcagg tggtaacgga 7260 gttggatact gctcgtgcgt gtccaggtcc atgctcctga aaatgaaggc acacattgac 7320 cctgaaccac accacgaggg gttgattgtt gataccagag atgtggaaga gcgcgtgcat 7380 gtcatgcgta aaaccaagct tgcacccacc gtggcacacg gtgtgtttaa ccctgaattt 7440 ggtcccgctg ccttgtccaa caaggacccg cggctgaacg aaggggttgt cctcgatgaa 7500 gtcatcttct ccaaacacaa gggagacacg aaaatgtctg aggaggacaa agcgctgttc 7560 cgccgctgcg ctgccgacta cgcgtcgcac ttgcacagcg tgctggggac ggcaaatgcc 7620 ccattgagca tctatgaggc catcaaaggc gtcgacgggc tcgatgccat ggagccggac 7680 accgcgcccg gcctcccctg ggccctccag gggaaacgcc gtggtgcgtt gattgacttc 7740 gagaacggca cggtcggacc cgaagtcgag gctgccctaa agctcatgga gaaaagagag 7800 tacaaatttg cttgccagac cttcctgaaa gacgagattc gtccgatgga aaaagtacgt 7860 gctggcaaga ctcgcattgt cgacgttttg cccgtggaac acattcttta caccaggatg 7920 atgattggca gattctgtgc tcaaatgcac acaaacaatg gaccgcagat tggctcagcg 7980 gtcggttgca atcctgatgt tgattggcaa agatttggca cacattttgc tcagtacaga 8040 aacgtgtggg atgtggacta ttcggccttt gatgctaacc actgcagtga cgcaatgaac 8100 atcatgtttg aggaggtatt tcgcacagac ttcggtttcc acccaaatgc tgagtggatt 8160 ctgaagactc ttgtgaacac ggagcacgcc tatgagaaca aacgtatcac tgttgagggc 8220 gggatgccgt ctggctgttc cgcgacaagc atcatcaaca caattttgaa caacatttat 8280 gtgctctacg ctcttcgtag acactatgag ggagttgagc tggacaccta caccatgatc 8340 tcctacggag atgacatcgt ggttgcaagt gactacgatc tggattttga ggctctcaaa 8400 ccccacttca aatctcttgg tcaaaccatc actccagctg acaaaagcga caaaggtttt 8460 gttcttggtc actccattac cgatgtcact ttcctcaaaa gacacttcca catggactat 8520 ggaactgggt tttacaaacc tgtgatggcc tcaaagaccc tcgaggccat tctctccttt 8580 gcacgccgtg ggaccataca ggagaagttg atctccgtgg caggactcgc cgtccactca 8640 ggacctgacg agtaccggcg tctctttgag cccttccagg gtctcttcga gattccaagc 8700 tacagatcac tttacctgcg ttgggtgaac gccgtgtgcg gtgacgcata atccctcaga 8760 tgtcacaatt ggcagaaaga ctctgaggcg agcgacgccg taggagtgaa aagcccgaaa 8820 gggcttttcc cgcttcctat tccaaaaaaa aaaaaaaaaa actagttcta gagcggccgc 8880 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt 8940 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 9000 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 9060 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 9120 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 9180 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 9240 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 9300 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 9360 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 9420 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 9480 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 9540 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct 9600 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 9660 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 9720 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 9780 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 9840 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 9900 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 9960 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 10020 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 10080 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 10140 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 10200 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 10260 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 10320 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 10380 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 10440 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 10500 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 10560 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 10620 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 10680 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 10740 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 10800 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 10860 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 10920 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 10980 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 11040 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac 11097 <210> 2 <211> 2205 <212> DNA <213> Artificial Sequence <220> <223> A22/Iraq/24/64 P1 (VP4-VP1) <400> 2 ggagccgggc aatccagtcc ggcgactggg tcgcagaacc agtcaggcaa cactgggagt 60 attattaaca actactacat gcaacagtac cagaactcca tggacaccca attaggtgac 120 aacgctataa gcggaggctc caatgaggga tccacggaca caacttccac ccacacaacc 180 aacactcaga acaacgactg gttttcaaag cttgccagtt ctgctttcag cggtcttttc 240 ggcgcccttc tcgccgataa gaagaccgag gagaccactc tcctcgagga ccgcattctc 300 accacccgca acgggcacac cacctccaca acccaatcga gtgtgggagt cacgtacggg 360 tactccaccc aggaagatca tgtttccgga cctaacacat ctggtttgga gacgcgggtg 420 gtgcaggcag aaagattttt caagaagcac ctgtttgatt ggacaccgga caaagctttt 480 gggcacttag agaagttgga acttcccact gaccacaagg gagtctacgg acacttggtg 540 gactcatttg catacatgag aaatggctgg gacgtggagg tgtccgctgt tggcaaccag 600 tttaacggcg ggtgtctcct ggtggccatg gtccctgaat ggaaagagtt caccccgcgt 660 gagaagtacc agctcacttt gtttccacac cagttcatca gccccagaac caacatgact 720 gcccacatcg tagtcccgta ccttggtgtg aacaggtacg accagtataa gaagcacaaa 780 ccctggacgc tggttgtgat ggtggtctca ccgctcacca ccaacactgt tagtgcagga 840 caaatcaagg tttatgccaa cattgccccg actcacgttc acgtggccgg cgagctcccc 900 tcgaaagagg ggattgtacc ggtcgcttgt tcggacgggt acggtggctt ggtgacaaca 960 gacccaaaaa cagctgaccc tgtttatggt atggtgtaca acccccccag gacaaactac 1020 cccgggcggt tcacaaacct gttggatgtg gctgaggcct gccccacctt tctctgtttc 1080 gacgaaggga aaccgtacgt tgtgacaaga acggacgagc agcgtcttct ggccaagttc 1140 gacgtctctc ttgctgcaaa gcacatgtca aacacctacc tttcagggat agcacagtac 1200 tacgcacagt actctggtac catcaacctg cacttcatgt ttaccggctc cacggattca 1260 aaagcccgct acatggtggc gtacgttcca cccggtgtgg agacaccgcc ggacacgcct 1320 gagaaagctg cacactgcat ccatgctgag tgggacacag ggttgaactc caagtttact 1380 ttctctatcc cgtacgtgtc tgccgcagat tacgcgtaca ctgcgtctga tgtggcagaa 1440 acaacaaacg tacagggatg ggtctgcata taccaaatta cacacgggaa agctgaacaa 1500 gacactctgg ttgtgtcggc tagcgccggc aaggactttg agttgcgcct cccgattgac 1560 ccccgctcac aaaccactac caccggggag tctgcagacc ctgtcaccac cactgttgaa 1620 aactacggcg gtgagacaca agtccaacga cgtcagcaca ccgacgttac tttcataatg 1680 gacagattg taaagataca aaacttgaac cccacacatg tcattgacct catgcaaacc 1740 caccaacacg ggttggtagg tgccctgtta cgtgctgcta cgtactactt ctctgacctg 1800 gagattgtgg tacgccatga cggtaaccta acctgggtac ccaatggagc acccgaggca 1860 gctctgtcta acacgggcaa ccccaccgcc tacctcaagg caccatttac gaggctcgcg 1920 ctcccctaca ccgcgccaca ccgcgtgttg gcaacagtgt acaacgggac gagcaagtac 1980 tccgcaggtg gtacgggcag acggggcgac ctagggcctc tcgcggcgag ggtcgccgct 2040 cagcttcctg cttctttcaa ctttggtgca attcaagcca cgaccatcca cgagctcctc 2100 gtgcgcatga agcgtgccga actctactgc cccagaccac tgttggcagt ggaggtgtcg 2160 tctcaagaca gacacaaaca gaagatcatt gcacctgcaa aacaa 2205 <210> 3 <211> 11163 <212> DNA <213> Artificial Sequence <220> <223> pOm-A22-P1 plasmid <400> 3 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 ccccctaggt tttaccgtcg ttcccgacgt taatggggaa acaaccacaa 1080 gcttaacacc gtcttgcccg acgtaaaagg gctgcaacca aaaagcttgt gccgcctttc 1140 ccggcgttaa tgggaggtaa ccacaagaca aaccttcacc cggaagtaaa acggcaactt 1200 cacacagttt tgcccgtttt cgtgagaaat gggccgtcaa cgcacgaaac gcgccgtcgc 1260 ttgaggagga cttgtacaaa cacgatctat gcaggtttcc acaactgaca caaaccgtgc 1320 aacttgaaac cccgcctggt ctttccaggt ctagaggggc gacattttgt actgtgcttg 1380 actccacgct cggtccacta gcgagtgtta gtagtagcac tgttgcttcg tagcggagca 1440 tgatggccgt gggagcttcc ccttggtaac aaggacccac ggggccaaaa gccacgtcct 1500 accggaccca tcatgtgtgc aaacccagca cggcaacttt actgcgaaaa ccactttaag 1560 gtgacactga tactggtact caatcactgg tgacaggcta aggatgccct tcaggtaccc 1620 cgaggtaaca cgcgacactc gggatctgag aaggggactg gggcttcttt aaaagtgccc 1680 agtttaaaaa gcttctatgc ctgaataggc gaccggaggc cggcgccttt tcactgtttt 1740 actactgttt tcatgaatac aactgactgt ttcaccgccc tgttacacgc tctcagagag 1800 atcaaaacac tgtttctttt acggacacaa ggaaagatgg aattcacact ttacaacggt 1860 gagaagaaaa ccttctactc cagacccaac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggtatgt tgatgagcct ttctttgact gggtctacga ctcgcctgaa 1980 aacctcactc ttgaggcaat caaacagttg gaagagacaa ccggtcttga gctgcacgag 2040 ggtggaccac ccgctctcgt catctggaac atcaaacact tgcttcacac cggaatcggc 2100 actgcctcac gccctagcga ggtgtgtatg gtggacggaa cggacatgtg tttagctgat 2160 tttcatgctg gcattttcct gaaaggacag gaacatgctg tgttcgcctg tgtcacctcc 2220 aacgggtggt acgcgattga tgacgaggac ttttaccctt ggacaccgga cccgtccgac 2280 gttctggtgt ttgtcccgta cgatcaagaa ccgcttaacg gagagtggaa aacaaaggtc 2340 cagaaaaggc tcaagggagc cgggcaatcc agtccggcga ctgggtcgca gaaccagtca 2400 ggcaacactg ggagtattat taacaactac tacatgcaac agtaccagaa ctccatggac 2460 acccaattag gtgacaacgc tataagcgga ggctccaatg agggatccac ggacacaact 2520 tccacccaca caaccaacac tcagaacaac gactggtttt caaagcttgc cagttctgct 2580 ttcagcggtc ttttcggcgc ccttctcgcc gataagaaga ccgaggagac cactctcctc 2640 gaggaccgca ttctcaccac ccgcaacggg cacaccacct ccacaaccca atcgagtgtg 2700 ggagtcacgt acgggtactc cacccaggaa gatcatgttt ccggacctaa cacatctggt 2760 ttggagacgc gggtggtgca ggcagaaaga tttttcaaga agcacctgtt tgattggaca 2820 ccggacaaag cttttgggca cttagagaag ttggaacttc ccactgacca caagggagtc 2880 tacggacact tggtggactc atttgcatac atgagaaatg gctgggacgt ggaggtgtcc 2940 gctgttggca accagtttaa cggcgggtgt ctcctggtgg ccatggtccc tgaatggaaa 3000 gagttcaccc cgcgtgagaa gtaccagctc actttgtttc cacaccagtt catcagcccc 3060 agaaccaaca tgactgccca catcgtagtc ccgtaccttg gtgtgaacag gtacgaccag 3120 tataagaagc acaaaccctg gacgctggtt gtgatggtgg tctcaccgct caccaccaac 3180 actgttagtg caggacaaat caaggtttat gccaacattg ccccgactca cgttcacgtg 3240 gccggcgagc tcccctcgaa agaggggatt gtaccggtcg cttgttcgga cgggtacggt 3300 ggcttggtga caacagaccc aaaaacagct gaccctgttt atggtatggt gtacaacccc 3360 cccaggacaa actaccccgg gcggttcaca aacctgttgg atgtggctga ggcctgcccc 3420 acctttctct gtttcgacga agggaaaccg tacgttgtga caagaacgga cgagcagcgt 3480 cttctggcca agttcgacgt ctctcttgct gcaaagcaca tgtcaaacac ctacctttca 3540 gggatagcac agtactacgc acagtactct ggtaccatca acctgcactt catgtttacc 3600 ggctccacgg attcaaaagc ccgctacat gtggcgtacg ttccacccgg tgtggagaca 3660 ccgccggaca cgcctgagaa agctgcacac tgcatccatg ctgagtggga cacagggttg 3720 aactccaagt ttactttctc tatcccgtac gtgtctgccg cagattacgc gtacactgcg 3780 tctgatgtgg cagaaacaac aaacgtacag ggatgggtct gcatatacca aattacacac 3840 gggaaagctg aacaagacac tctggttgtg tcggctagcg ccggcaagga ctttgagttg 3900 cgcctcccga ttgacccccg ctcacaaacc actaccaccg gggagtctgc agaccctgtc 3960 accaccactg ttgaaaacta cggcggtgag acacaagtcc aacgacgtca gcacaccgac 4020 gttactttca taatggacag atttgtaaag atacaaaact tgaaccccac acatgtcatt 4080 gacctcatgc aaacccacca acacgggttg gtaggtgccc tgttacgtgc tgctacgtac 4140 tacttctctg acctggagat tgtggtacgc catgacggta acctaacctg ggtacccaat 4200 ggagcacccg aggcagctct gtctaacacg ggcaacccca ccgcctacct caaggcacca 4260 tttacgaggc tcgcgctccc ctacaccgcg ccacaccgcg tgttggcaac agtgtacaac 4320 gggacgagca agtactccgc aggtggtacg ggcagacggg gcgacctagg gcctctcgcg 4380 caaccgtcgg tgcagatcca ggtctatcag ggggagcgtg agatcgccgc gcacaacaag 4440 gcgagggtcg ccgctcagct tcctgcttct ttcaactttg gtgcaattca agccacgacc 4500 atccacgagc tcctcgtgcg catgaagcgt gccgaactct actgccccag accactgttg 4560 gcagtggagg tgtcgtctca agacagacac aaacagaaga tcattgcacc tgcaaaacaa 4620 cttctaaatt ttgacctgct caaattggcg ggagatgtgg agtccaaccc tgggcccttc 4680 ttcttctccg acgtcaggtc aaatttctca aaactggtag aaaccatcaa tcagatgcag 4740 gaggacatgt caacaaaaca cgggcctgac tttaaccggt tggtgtccgc atttgaggaa 4800 ttggccactg gagtgaaggc tatcagggcc ggtctcgacg aggccaaacc ctggtacaaa 4860 ctcatcaagc tcctgagccg cttgtcgtgc atggccgctg tagcagcacg gtcaaaggac 4920 ccagtccttg tggccatcat gctggctgac accggtcttg agattctgga cagcaccttt 4980 gtcgtgaaga agatctccga ctcgctctcc agtctctttc acgtgccggc ccccgtcttc 5040 agtttcggag ccccgattct gttggccggg ttggtcaaag tcgcctcgag tttcttccgg 5100 tccacacccg aagaccttga gagagcagaa aaacagctca aagcacgtga cattaacgac 5160 atattcgcca ttctcaagaa cggcgagtgg ctggtcaagc tgatccttgc catccgcgac 5220 tggatcaaag cgtggatcgc ctcagaagaa aagtttgtca ccatgacgga cttggtgcct 5280 ggtatccttg aaaagcagcg ggatctcaac gacccgagta agtacaagga agccaaggag 5340 tggctcgaca acgcgcgcca ggcgtgtttg aagagcggga acgttcacat tgccaatttg 5400 tgcaaagtgg tcgccccggc acccagcaag tcgagacccg aacccgtggt cgtttgcctc 5460 cgcggcaaat ccggccaggg gaagagtttc cttgcgaacg tgctcgcgca agcaatctcc 5520 acccacttca ccggcagaac tgattcggtt tggtactgcc cgcctgaccc tgaccacttc 5580 gacggttaca accagcagac cgttgtcgtg atggacgatt tgggccagaa ccccgatggc 5640 aaggacttca agtacttcgc ccagatggtt tcgaccacgg ggttcatccc gcccatggcc 5700 tcgcttgagg acaaaggcaa gcctttcaac agcaaagtca tcattgctac caccaacctg 5760 tactcgggtt tcaccccgag aacaatggtg tgtcctgacg cgctgaaccg gaggttccac 5820 tttgacatcg acgtgagtgc caaggacggg tacaaagtta acaacaaatt ggacataatc 5880 aaagctcttg aagacaccca caccaaccca gtggcgatgt tccaatacga ctgtgccctt 5940 ctaaacggta tggcagttga aatgaagaga atgcaacagg atatgttcaa gcctcaacca 6000 cccctccaga acgtgtacca actcgttcac gaggtgattg aacgggtcga gctccacgag 6060 aaggtgtcga gccacccgat tttcaaacag atatcaattc cttcccaaaa gtctgtgttg 6120 tacttcctca ttgagaaagg ccaacacgaa gcagcaattg aattctttga gggaatggtg 6180 catgactcca tcaaggaaga gctccggccc ctcatccaac agacctcatt tgtgaaacgc 6240 gcttttaagc gcctgaagga aaactttgag actgttgccc tgtgtttgac tcttttggca 6300 aacatagtga tcatgatccg cgagactcgc aagagacaac agatggtgga cgatgcagtg 6360 aatgactaca ttgagaaggc aaacatcacc acagatgaca agactcttga cgaggcggaa 6420 aagaaccctc tagagaccag cggtgccagc actattggtt tcagagagag aactctcccg 6480 gggcacaagg cgagcgatga cgtgagctcc gagcccgcca aacccgtgga ggaccgacca 6540 caagctgaag gaccttacga gggaccggtg aagaagcctg tcgctttgaa agtgaaagct 6600 aagaacttga ttgtcactga ggggccatat gaaggaccag tgaagaaacc tgtcgctttg 6660 aaagtgaaag caaaagcccc gattgtcact gaaggaccct acgagggacc ggtgaagaag 6720 cctgtcgctt tgaaagtgaa agccaagaac ttgattgtca ctgagagtgg tgccccaccg 6780 accgacttgc agaagatggt catgggcaac actaagcctg ttgagctcat cctcgagggg 6840 aagacggtag ccatctgctg tgctaccgga gtgtttggca ctgcctacct cgtacctcgt 6900 cacctcttcg cggagaagta cgacaagata atgttggacg gtagagccat gacagacagt 6960 gactacagag tgtttgagtt tgagattaaa gtaaaaggac aggacatgct ctcagacgct 7020 gcactcatgg tgcttcaccg tgggaaccgc gtgagagaca tcacgaaaca ttttcgtgac 7080 acagcaagaa tgaagaaagg cacccccgtt gtcggtgtga tcaacaacgc cgacgttggg 7140 agactgattt tctctggaga ggcccttacc tacaaagaca ttgtagtgac catggatgga 7200 gacaccatgc cgggcctgtt tgcctacaga gccgccacca aggctggtta ctgcggggga 7260 gccgttctcg ccaaggacgg agccgacaca ttcatcgttg gcacccactc cgcaggtggt 7320 aacggagttg gatactgctc gtgcgtgtcc aggtccatgc tcctgaaaat gaaggcacac 7380 attgaccctg aaccacacca cgaggggttg attgttgata ccagagatgt ggaagagcgc 7440 gtgcatgtca tgcgtaaaac caagcttgca cccaccgtgg cacacggtgt gtttaaccct 7500 gaatttggtc ccgctgcctt gtccaacaag gacccgcggc tgaacgaagg ggttgtcctc 7560 gatgaagtca tcttctccaa acacaaggga gacacgaaaa tgtctgagga ggacaaagcg 7620 ctgttccgcc gctgcgctgc cgactacgcg tcgcacttgc acagcgtgct ggggacggca 7680 aatgccccat tgagcatcta tgaggccatc aaaggcgtcg acgggctcga tgccatggag 7740 ccggacaccg cgcccggcct cccctgggcc ctccagggga aacgccgtgg tgcgttgatt 7800 gacttcgaga acggcacggt cggacccgaa gtcgaggctg ccctaaagct catggagaaa 7860 agagagtaca aatttgcttg ccagaccttc ctgaaagacg agattcgtcc gatggaaaaa 7920 gtacgtgctg gcaagactcg cattgtcgac gttttgcccg tggaacacat tctttacacc 7980 aggatgatga ttggcagatt ctgtgctcaa atgcacacaa acaatggacc gcagattggc 8040 tcagcggtcg gttgcaatcc tgatgttgat tggcaaagat ttggcacaca ttttgctcag 8100 tacagaaacg tgtgggatgt ggactattcg gcctttgatg ctaaccactg cagtgacgca 8160 atgaacatca tgtttgagga ggtatttcgc acagacttcg gtttccaccc aaatgctgag 8220 tggattctga agactcttgt gaacacggag cacgcctatg agaacaaacg tatcactgtt 8280 gagggcggga tgccgtctgg ctgttccgcg acaagcatca tcaacacaat tttgaacaac 8340 atttatgtgc tctacgctct tcgtagacac tatgagggag ttgagctgga cacctacacc 8400 atgatctcct acggagatga catcgtggtt gcaagtgact acgatctgga ttttgaggct 8460 ctcaaacccc acttcaaatc tcttggtcaa accatcactc cagctgacaa aagcgacaaa 8520 ggttttgttc ttggtcactc cattaccgat gtcactttcc tcaaaagaca cttccacatg 8580 gactatggaa ctgggtttta caaacctgtg atggcctcaa agaccctcga ggccattctc 8640 tcctttgcac gccgtgggac catacaggag aagttgatct ccgtggcagg actcgccgtc 8700 cactcaggac ctgacgagta ccggcgtctc tttgagccct tccagggtct cttcgagatt 8760 ccaagctaca gatcacttta cctgcgttgg gtgaacgccg tgtgcggtga cgcataatcc 8820 ctcagatgtc acaattggca gaaagactct gaggcgagcg acgccgtagg agtgaaaagc 8880 ccgaaagggc ttttcccgct tcctattcca aaaaaaaaaa aaaaaaacta gttctagagc 8940 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct 9000 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 9060 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 9120 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 9180 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 9240 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 9300 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 9360 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 9420 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 9480 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 9540 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 9600 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 9660 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 9720 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 9780 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 9840 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 9900 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 9960 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 10020 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 10080 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 10140 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 10200 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 10260 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 10320 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 10380 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 10440 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 10500 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 10560 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 10620 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 10680 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 10740 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 10800 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 10860 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 10920 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 10980 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 11040 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 11100 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 11160 cac 11163 <210> 4 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> HSP70 <400> 4 caaccgtcgg tgcagatcca ggtctatcag ggggagcgtg agatcgccgc gcacaacaag 60 60 <210> 5 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> HSP70 <400> 5 Gln Pro Ser Val Gln Ile Gln Val Tyr Gln Gly Glu Arg Glu Ile Ala 1 5 10 15 Ala His Asn Lys 20 <210> 6 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for HSP70 <400> 6 ggggagcgtg agatcgccgc gcacaacaag gcgagggtcg ccgctcagct 50 <210> 7 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for HSP70 <400> 7 ctgatagacc tggatctgca ccgacggttg cgcgagaggc cctaggtcgc 50 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> forward universal primer VP1 <400> 8 aggcnggna artttga 17 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse universal primer VP1 <400> 9 catgtcntcc atctggtt 18 <210> 10 <211> 11163 <212> DNA <213> Artificial Sequence <220> <223> pOm-A22-P1-HSP70 <400> 10 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 ccccctaggt tttaccgtcg ttcccgacgt taatggggaa acaaccacaa 1080 gcttaacacc gtcttgcccg acgtaaaagg gctgcaacca aaaagcttgt gccgcctttc 1140 ccggcgttaa tgggaggtaa ccacaagaca aaccttcacc cggaagtaaa acggcaactt 1200 cacacagttt tgcccgtttt cgtgagaaat gggccgtcaa cgcacgaaac gcgccgtcgc 1260 ttgaggagga cttgtacaaa cacgatctat gcaggtttcc acaactgaca caaaccgtgc 1320 aacttgaaac cccgcctggt ctttccaggt ctagaggggc gacattttgt actgtgcttg 1380 actccacgct cggtccacta gcgagtgtta gtagtagcac tgttgcttcg tagcggagca 1440 tgatggccgt gggagcttcc ccttggtaac aaggacccac ggggccaaaa gccacgtcct 1500 accggaccca tcatgtgtgc aaacccagca cggcaacttt actgcgaaaa ccactttaag 1560 gtgacactga tactggtact caatcactgg tgacaggcta aggatgccct tcaggtaccc 1620 cgaggtaaca cgcgacactc gggatctgag aaggggactg gggcttcttt aaaagtgccc 1680 agtttaaaaa gcttctatgc ctgaataggc gaccggaggc cggcgccttt tcactgtttt 1740 actactgttt tcatgaatac aactgactgt ttcaccgccc tgttacacgc tctcagagag 1800 atcaaaacac tgtttctttt acggacacaa ggaaagatgg aattcacact ttacaacggt 1860 gagaagaaaa ccttctactc cagacccaac aaccacgaca actgctggct taacaccatt 1920 ctccagttgt tcaggtatgt tgatgagcct ttctttgact gggtctacga ctcgcctgaa 1980 aacctcactc ttgaggcaat caaacagttg gaagagacaa ccggtcttga gctgcacgag 2040 ggtggaccac ccgctctcgt catctggaac atcaaacact tgcttcacac cggaatcggc 2100 actgcctcac gccctagcga ggtgtgtatg gtggacggaa cggacatgtg tttagctgat 2160 tttcatgctg gcattttcct gaaaggacag gaacatgctg tgttcgcctg tgtcacctcc 2220 aacgggtggt acgcgattga tgacgaggac ttttaccctt ggacaccgga cccgtccgac 2280 gttctggtgt ttgtcccgta cgatcaagaa ccgcttaacg gagagtggaa aacaaaggtc 2340 cagaaaaggc tcaagggagc cgggcaatcc agtccggcga ctgggtcgca gaaccagtca 2400 ggcaacactg ggagtattat taacaactac tacatgcaac agtaccagaa ctccatggac 2460 acccaattag gtgacaacgc tataagcgga ggctccaatg agggatccac ggacacaact 2520 tccacccaca caaccaacac tcagaacaac gactggtttt caaagcttgc cagttctgct 2580 ttcagcggtc ttttcggcgc ccttctcgcc gataagaaga ccgaggagac cactctcctc 2640 gaggaccgca ttctcaccac ccgcaacggg cacaccacct ccacaaccca atcgagtgtg 2700 ggagtcacgt acgggtactc cacccaggaa gatcatgttt ccggacctaa cacatctggt 2760 ttggagacgc gggtggtgca ggcagaaaga tttttcaaga agcacctgtt tgattggaca 2820 ccggacaaag cttttgggca cttagagaag ttggaacttc ccactgacca caagggagtc 2880 tacggacact tggtggactc atttgcatac atgagaaatg gctgggacgt ggaggtgtcc 2940 gctgttggca accagtttaa cggcgggtgt ctcctggtgg ccatggtccc tgaatggaaa 3000 gagttcaccc cgcgtgagaa gtaccagctc actttgtttc cacaccagtt catcagcccc 3060 agaaccaaca tgactgccca catcgtagtc ccgtaccttg gtgtgaacag gtacgaccag 3120 tataagaagc acaaaccctg gacgctggtt gtgatggtgg tctcaccgct caccaccaac 3180 actgttagtg caggacaaat caaggtttat gccaacattg ccccgactca cgttcacgtg 3240 gccggcgagc tcccctcgaa agaggggatt gtaccggtcg cttgttcgga cgggtacggt 3300 ggcttggtga caacagaccc aaaaacagct gaccctgttt atggtatggt gtacaacccc 3360 cccaggacaa actaccccgg gcggttcaca aacctgttgg atgtggctga ggcctgcccc 3420 acctttctct gtttcgacga agggaaaccg tacgttgtga caagaacgga cgagcagcgt 3480 cttctggcca agttcgacgt ctctcttgct gcaaagcaca tgtcaaacac ctacctttca 3540 gggatagcac agtactacgc acagtactct ggtaccatca acctgcactt catgtttacc 3600 ggctccacgg attcaaaagc ccgctacat gtggcgtacg ttccacccgg tgtggagaca 3660 ccgccggaca cgcctgagaa agctgcacac tgcatccatg ctgagtggga cacagggttg 3720 aactccaagt ttactttctc tatcccgtac gtgtctgccg cagattacgc gtacactgcg 3780 tctgatgtgg cagaaacaac aaacgtacag ggatgggtct gcatatacca aattacacac 3840 gggaaagctg aacaagacac tctggttgtg tcggctagcg ccggcaagga ctttgagttg 3900 cgcctcccga ttgacccccg ctcacaaacc actaccaccg gggagtctgc agaccctgtc 3960 accaccactg ttgaaaacta cggcggtgag acacaagtcc aacgacgtca gcacaccgac 4020 gttactttca taatggacag atttgtaaag atacaaaact tgaaccccac acatgtcatt 4080 gacctcatgc aaacccacca acacgggttg gtaggtgccc tgttacgtgc tgctacgtac 4140 tacttctctg acctggagat tgtggtacgc catgacggta acctaacctg ggtacccaat 4200 ggagcacccg aggcagctct gtctaacacg ggcaacccca ccgcctacct caaggcacca 4260 tttacgaggc tcgcgctccc ctacaccgcg ccacaccgcg tgttggcaac agtgtacaac 4320 gggacgagca agtactccgc aggtggtacg ggcagacggg gcgacctagg gcctctcgcg 4380 caaccgtcgg tgcagatcca ggtctatcag ggggagcgtg agatcgccgc gcacaacaag 4440 gcgagggtcg ccgctcagct tcctgcttct ttcaactttg gtgcaattca agccacgacc 4500 atccacgagc tcctcgtgcg catgaagcgt gccgaactct actgccccag accactgttg 4560 gcagtggagg tgtcgtctca agacagacac aaacagaaga tcattgcacc tgcaaaacaa 4620 cttctaaatt ttgacctgct caaattggcg ggagatgtgg agtccaaccc tgggcccttc 4680 ttcttctccg acgtcaggtc aaatttctca aaactggtag aaaccatcaa tcagatgcag 4740 gaggacatgt caacaaaaca cgggcctgac tttaaccggt tggtgtccgc atttgaggaa 4800 ttggccactg gagtgaaggc tatcagggcc ggtctcgacg aggccaaacc ctggtacaaa 4860 ctcatcaagc tcctgagccg cttgtcgtgc atggccgctg tagcagcacg gtcaaaggac 4920 ccagtccttg tggccatcat gctggctgac accggtcttg agattctgga cagcaccttt 4980 gtcgtgaaga agatctccga ctcgctctcc agtctctttc acgtgccggc ccccgtcttc 5040 agtttcggag ccccgattct gttggccggg ttggtcaaag tcgcctcgag tttcttccgg 5100 tccacacccg aagaccttga gagagcagaa aaacagctca aagcacgtga cattaacgac 5160 atattcgcca ttctcaagaa cggcgagtgg ctggtcaagc tgatccttgc catccgcgac 5220 tggatcaaag cgtggatcgc ctcagaagaa aagtttgtca ccatgacgga cttggtgcct 5280 ggtatccttg aaaagcagcg ggatctcaac gacccgagta agtacaagga agccaaggag 5340 tggctcgaca acgcgcgcca ggcgtgtttg aagagcggga acgttcacat tgccaatttg 5400 tgcaaagtgg tcgccccggc acccagcaag tcgagacccg aacccgtggt cgtttgcctc 5460 cgcggcaaat ccggccaggg gaagagtttc cttgcgaacg tgctcgcgca agcaatctcc 5520 acccacttca ccggcagaac tgattcggtt tggtactgcc cgcctgaccc tgaccacttc 5580 gacggttaca accagcagac cgttgtcgtg atggacgatt tgggccagaa ccccgatggc 5640 aaggacttca agtacttcgc ccagatggtt tcgaccacgg ggttcatccc gcccatggcc 5700 tcgcttgagg acaaaggcaa gcctttcaac agcaaagtca tcattgctac caccaacctg 5760 tactcgggtt tcaccccgag aacaatggtg tgtcctgacg cgctgaaccg gaggttccac 5820 tttgacatcg acgtgagtgc caaggacggg tacaaagtta acaacaaatt ggacataatc 5880 aaagctcttg aagacaccca caccaaccca gtggcgatgt tccaatacga ctgtgccctt 5940 ctaaacggta tggcagttga aatgaagaga atgcaacagg atatgttcaa gcctcaacca 6000 cccctccaga acgtgtacca actcgttcac gaggtgattg aacgggtcga gctccacgag 6060 aaggtgtcga gccacccgat tttcaaacag atatcaattc cttcccaaaa gtctgtgttg 6120 tacttcctca ttgagaaagg ccaacacgaa gcagcaattg aattctttga gggaatggtg 6180 catgactcca tcaaggaaga gctccggccc ctcatccaac agacctcatt tgtgaaacgc 6240 gcttttaagc gcctgaagga aaactttgag actgttgccc tgtgtttgac tcttttggca 6300 aacatagtga tcatgatccg cgagactcgc aagagacaac agatggtgga cgatgcagtg 6360 aatgactaca ttgagaaggc aaacatcacc acagatgaca agactcttga cgaggcggaa 6420 aagaaccctc tagagaccag cggtgccagc actattggtt tcagagagag aactctcccg 6480 gggcacaagg cgagcgatga cgtgagctcc gagcccgcca aacccgtgga ggaccgacca 6540 caagctgaag gaccttacga gggaccggtg aagaagcctg tcgctttgaa agtgaaagct 6600 aagaacttga ttgtcactga ggggccatat gaaggaccag tgaagaaacc tgtcgctttg 6660 aaagtgaaag caaaagcccc gattgtcact gaaggaccct acgagggacc ggtgaagaag 6720 cctgtcgctt tgaaagtgaa agccaagaac ttgattgtca ctgagagtgg tgccccaccg 6780 accgacttgc agaagatggt catgggcaac actaagcctg ttgagctcat cctcgagggg 6840 aagacggtag ccatctgctg tgctaccgga gtgtttggca ctgcctacct cgtacctcgt 6900 cacctcttcg cggagaagta cgacaagata atgttggacg gtagagccat gacagacagt 6960 gactacagag tgtttgagtt tgagattaaa gtaaaaggac aggacatgct ctcagacgct 7020 gcactcatgg tgcttcaccg tgggaaccgc gtgagagaca tcacgaaaca ttttcgtgac 7080 acagcaagaa tgaagaaagg cacccccgtt gtcggtgtga tcaacaacgc cgacgttggg 7140 agactgattt tctctggaga ggcccttacc tacaaagaca ttgtagtgac catggatgga 7200 gacaccatgc cgggcctgtt tgcctacaga gccgccacca aggctggtta ctgcggggga 7260 gccgttctcg ccaaggacgg agccgacaca ttcatcgttg gcacccactc cgcaggtggt 7320 aacggagttg gatactgctc gtgcgtgtcc aggtccatgc tcctgaaaat gaaggcacac 7380 attgaccctg aaccacacca cgaggggttg attgttgata ccagagatgt ggaagagcgc 7440 gtgcatgtca tgcgtaaaac caagcttgca cccaccgtgg cacacggtgt gtttaaccct 7500 gaatttggtc ccgctgcctt gtccaacaag gacccgcggc tgaacgaagg ggttgtcctc 7560 gatgaagtca tcttctccaa acacaaggga gacacgaaaa tgtctgagga ggacaaagcg 7620 ctgttccgcc gctgcgctgc cgactacgcg tcgcacttgc acagcgtgct ggggacggca 7680 aatgccccat tgagcatcta tgaggccatc aaaggcgtcg acgggctcga tgccatggag 7740 ccggacaccg cgcccggcct cccctgggcc ctccagggga aacgccgtgg tgcgttgatt 7800 gacttcgaga acggcacggt cggacccgaa gtcgaggctg ccctaaagct catggagaaa 7860 agagagtaca aatttgcttg ccagaccttc ctgaaagacg agattcgtcc gatggaaaaa 7920 gtacgtgctg gcaagactcg cattgtcgac gttttgcccg tggaacacat tctttacacc 7980 aggatgatga ttggcagatt ctgtgctcaa atgcacacaa acaatggacc gcagattggc 8040 tcagcggtcg gttgcaatcc tgatgttgat tggcaaagat ttggcacaca ttttgctcag 8100 tacagaaacg tgtgggatgt ggactattcg gcctttgatg ctaaccactg cagtgacgca 8160 atgaacatca tgtttgagga ggtatttcgc acagacttcg gtttccaccc aaatgctgag 8220 tggattctga agactcttgt gaacacggag cacgcctatg agaacaaacg tatcactgtt 8280 gagggcggga tgccgtctgg ctgttccgcg acaagcatca tcaacacaat tttgaacaac 8340 atttatgtgc tctacgctct tcgtagacac tatgagggag ttgagctgga cacctacacc 8400 atgatctcct acggagatga catcgtggtt gcaagtgact acgatctgga ttttgaggct 8460 ctcaaacccc acttcaaatc tcttggtcaa accatcactc cagctgacaa aagcgacaaa 8520 ggttttgttc ttggtcactc cattaccgat gtcactttcc tcaaaagaca cttccacatg 8580 gactatggaa ctgggtttta caaacctgtg atggcctcaa agaccctcga ggccattctc 8640 tcctttgcac gccgtgggac catacaggag aagttgatct ccgtggcagg actcgccgtc 8700 cactcaggac ctgacgagta ccggcgtctc tttgagccct tccagggtct cttcgagatt 8760 ccaagctaca gatcacttta cctgcgttgg gtgaacgccg tgtgcggtga cgcataatcc 8820 ctcagatgtc acaattggca gaaagactct gaggcgagcg acgccgtagg agtgaaaagc 8880 ccgaaagggc ttttcccgct tcctattcca aaaaaaaaaa aaaaaaacta gttctagagc 8940 ggccgccacc gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct 9000 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 9060 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 9120 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 9180 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 9240 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 9300 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 9360 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 9420 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 9480 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 9540 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 9600 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 9660 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 9720 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 9780 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 9840 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 9900 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 9960 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 10020 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 10080 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 10140 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 10200 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 10260 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 10320 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 10380 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 10440 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 10500 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 10560 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 10620 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 10680 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 10740 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 10800 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 10860 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 10920 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 10980 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 11040 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 11100 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 11160 cac 11163

Claims (8)

A recombinant plasmid having the sequence of SEQ ID NO:10. A recombinant foot-and-mouth disease virus obtained by culturing after transducing the recombinant plasmid of claim 1 into cells. A foot-and-mouth disease virus serotype antigen obtained by purifying and isolating the recombinant foot-and-mouth disease virus of claim 2. A vaccine composition comprising the recombinant foot-and-mouth disease virus of claim 2 or the purified, isolated foot-and-mouth disease virus A serotype antigen of claim 3. 5. The vaccine composition according to claim 4, wherein the vaccine composition is administered at a dose of 1/40 to 1/10 dose of the recombinant FMD virus or purified, isolated FMD virus A serotype antigen. A foot-and-mouth disease diagnostic kit comprising the foot-and-mouth disease recombinant virus of claim 2 or the foot-and-mouth disease virus A serotype antigen of claim 3. A method for diagnosing foot-and-mouth disease in animals other than humans using the foot-and-mouth disease diagnostic kit of claim 6. A method for preventing and treating foot-and-mouth disease in animals other than humans by using the vaccine composition comprising the recombinant virus of claim 2 or the antigen of the foot-and-mouth disease virus A serotype of claim 3.

Images