KR101578425B1 - Foot-and-mouth disease virus expressing P1-protective antigen of SAT1-WZ topotype and the manufacturing methods - Google Patents

Abstract

The present invention relates to a gene encoding a P1 protein, which is a protective antigen of the foot-and-mouth disease type O vaccine Manisa, of the whole gene region of a foot-and-mouth disease O-type vaccine strain Manisa is a gene encoding a P1 protein which is a defense antigen of foot- A recombinant foot-and-mouth disease virus comprising a plasmid in which a substituted recombinant gene is inserted, and a method for producing the same.

Description

Field of the Invention The present invention relates to a recombinant virus expressing a foot-and-mouth disease virus expressing a SAT1-type WZ-type protective antigen and a method for producing the same.

The present invention relates to recombinant foot-and-mouth disease virus (VZV) expressing a protective antigen of WZ type virus among various regional types of foot-and-mouth disease type SAT1, and more particularly to a recombinant foot- A recombinant foot-and-mouth disease virus comprising a plasmid into which a recombinant gene substituted with a gene encoding a P1 protein, which is a defense antigen of foot-and-mouth disease SAT1 type WZ locus virus, ≪ / RTI >

Foot-and-mouth disease (FMD) is a virus-borne disease that infects animals with split hoofs and is characterized by rapid replication and rapid spreading. Due to its economic importance, this disease is classified as a very important animal disease that can be transmitted by the World Animal Health Organization (OIE). Due to these characteristics, the occurrence of livestock products in the international trade is the most important check factor .

The foot-and-mouth disease pathogen is a single-stranded bipolar RNA virus, which belongs to the genus Piconaviridae and Aphthovirus and has seven different serotypes (A, O, C, Asia, SAT 1, SAT 2 and SAT 3) Being classified.

The foot-and-mouth disease virus of the SAT has been genetically altered to have a very diverse and regional character, and is highly antigenic. These virus serotypes are characterized by the ability to acquire the ability to infect multiple wildlife, depending on local circumstances. African bison in South Africa is susceptible to infection with SAT viruses and may also act as an important propagation factor for livestock infection.

Buffalo is not well-documented clinical symptoms in foot-and-mouth disease and may also function as a carrier. Foot-and-mouth disease has been reported in South Africa since 2000, with most cases occurring in cattle in sub-Saharan Africa. . In the case of SAT1, it is spread widely, and in most African buffalo, there is a persistent infection at 55%. On the other hand, SAT3 has the lowest incidence rate.

There are nine regional types of SAT1, I, II (SEZ), III (WZ), IV, V, VI, VII, VIII and IX. If strictly developed, vaccines for these local types should be developed and used as vaccines. However, for the development of all the vaccine strains, the outbreaks must be continuously cultured in cells to have adequate propagation capacity. However, this method is time consuming and there is no guarantee that the results will be good.

Foot-and-mouth disease viruses are also pathogenic agents in animals. It is internationally forbidden to be used as an attenuated vaccine, which has so far been reduced in pathogenicity to foot-and-mouth disease virus, since pathogenicity is maintained even in the attempts of various researchers such as multiple passages.

The use of attenuated virus against foot-and-mouth disease has a problem of restoration of virulence, and it is difficult to distinguish it from outbreaks of foot-and-mouth disease virus using live monsoon vaccine.

Therefore, it should be made into a form that can be produced in a timely manner so that a vaccine strain required by the existing vaccine virus can be rapidly developed and used.

Since the SAT1 serotype has nine local types with different characteristics, if the vaccine is developed considering the use in the field, it is necessary to develop all the vaccines against the local type or genotype and use it as a vaccine. To develop all the vaccines, Should be continuously cultured in cells to have adequate propagation capacity.

However, this method is time consuming and there is no guarantee that the results will be good. Therefore, it should be made into a form that can be produced in a timely manner so that a vaccine strain required by the existing vaccine virus can be rapidly developed and used.

In order to adequately defend against the SAT1 type WZ locus type, the present inventors have proposed a method of expressing a protective protein capable of defending against the WZ local virus based on a viral vector obtained by cloning the entire genome of foot-and-mouth disease virus O manisa vaccine into a plasmid And can provide a new recombinant foot-and-mouth disease virus. Thus, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a vaccine for a vaccine strain that can protect against the foot-and-mouth disease type SAT1 type WZ region, which is widely prevalent in the Middle East, by inserting P1 gene of foot-and-mouth disease virus SAT1 type into O manisa foot- RTI ID = 0.0 > foot < / RTI >

Another object of the present invention is to provide a method for producing recombinant foot-and-mouth disease virus which is capable of protecting against foot-and-mouth disease type SAT1 type WZ locomotives prevalent in Africa, and which can be usefully used as vaccine seedlings.

In order to achieve the above object, the present invention provides a gene encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as at least the protective antigen of the foot-and-mouth disease type O vaccine Manisa in the whole gene region of the foot- , A recombinant gene of SEQ ID NO: 1 substituted with a gene encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as a defensive antigen of the foot-and-mouth type SAT1 type WZ locus having the nucleotide sequence of Gene Berkman Session No. AY593842 A recombinant foot-and-mouth disease virus comprising a plasmid.

The present invention also relates to a method for producing a recombinant O-type vaccine strain (1) comprising the steps of: (1) inserting a whole gene of a foot-and-mouth type O-type vaccine strain Manisa into a plasmid; (2) VP1, VP2, VP3 and VP4 as protective antigens of Manisa were transfected with VP1, VP2, VP3 and VP4 as protective antigen of foot-and-mouth type SAT1 type WZ locus having the nucleotide sequence of GenBank accession session number AY593842 , And (3) introducing the recombinant plasmid constructed in the above step (2) into a cell to proliferate the recombinant plasmid. A method for producing recombinant foot-and mouth disease virus.

The recombinant foot-and-mouth disease virus according to the present invention has an advantage that it can be used more safely than a pathogenic virus because it is treated in a laboratory such as a disinfectant experiment or an antiviral experiment by weakening the pathogenicity to an animal by manipulating a part of a gene of foot-and-

In addition, the recombinant foot-and-mouth disease virus according to the present invention can effectively prevent the viruses occurring in Europe-South America region without showing significant difference between the virus titer and the original virus.

1 shows a schematic diagram of a plasmid (pO-Manisa) into which a whole gene of a foot-and-mouth disease type O vaccine strain Manisa is inserted.
2 is a schematic diagram of genome of recombinant FMD virus expressing a defensive antigen SAT1 type WZ region type protective antigen.
FIG. 3 is a schematic diagram of a recombinant plasmid (pOm-SAT1-SA-P1) in which a defensive protein gene of foot-and-mouth disease type SAT 1 WZ region vaccine is inserted.
FIG. 4 is a photograph showing a plaque formed in a small intestine cell (LF-BK) of a virus recovered from a plasmid in which a defensive protein gene of foot-and-mouth disease type SAT1 type WZ region is inserted.
FIG. 5 shows that a protein expressed in recombinant foot-and-mouth disease virus according to the present invention shows a foot-and-mouth antigen positive reaction using a liver antigen kit.
6 shows that recombinant foot-and-mouth disease virus (BFR) according to the present invention inhibits the expression of recombinant FMDVs in young hamster kidney cells (BHK21), black goat kidney cells (BGK), porcine kidney cells (IBRS- -BK) to form virus titer.

The present invention relates to a gene encoding a P1 protein comprising at least VP1, VP2, VP3 and VP4 as a protective antigen of at least the foot-and-mouth disease type O vaccine Manisa in the whole gene region of a foot-and-mouth disease type O vaccine strain Manisa, A recombinant foot-and-mouth disease virus comprising a plasmid in which a recombinant gene substituted with a gene encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as a defensive antigen SAT1 type WZ locus having a nucleotide sequence of AY593842 .

The SAT1 / 5sa / 61 strain of foot-and-mouth disease virus SAT1 can be protected against viruses in the WZ (Western Zimbabwe) region type, so the SAT1 / 5sa / 61 strain can be stored in the antigen bank Is one of the South African virus states.

Even if there is no current occurrence in Korea, if the SAT1 type is stored as an antigen bank in case of future occurrence, it can be unexpectedly defended when the SAT1 type foot-and-mouth disease develops. Especially, It can be useful.

In the present invention, a structural protein gene for SAT1 type WZ region type is inserted based on a viral vector obtained by cloning the whole genome of O1 manisa vaccine virus into a plasmid, and the protein is replaced with only the structural protein To create a new recombinant virus that expresses a protective protein that can protect against the WZ local virus.

In the recombinant FMD virus of the present invention, the plasmid into which the recombinant gene is inserted has the nucleotide sequence of SEQ ID NO: 1.

In the recombinant foot-and-mouth disease virus of the present invention, the recombinant virus is characterized by expressing a defensive antigen of the foot-and-mouth disease type SAT 1 type WZ region.

In the recombinant foot-and-mouth disease virus of the present invention, the recombinant virus is characterized by being a foot-and-mouth disease-backing virus.

The present invention also relates to a method for producing a recombinant O-type vaccine strain (1) comprising the steps of: (1) inserting a whole gene of a foot-and-mouth type O-type vaccine strain Manisa into a plasmid; (2) VP1, VP2, VP3 and VP4 as protective antigens of Manisa were transfected with VP1, VP2, VP3 and VP4 as protective antigen of foot-and-mouth type SAT1 type WZ locus having the nucleotide sequence of GenBank accession session number AY593842 , And (3) introducing the recombinant plasmid constructed in the above step (2) into a cell to proliferate the recombinant plasmid. To a method for producing recombinant foot-and mouth disease virus.

The step of inserting the whole gene of the foot-and-mouth disease type O vaccine strain Manisa into the plasmid may be performed by a method of inserting the gene into the plasmid, so that detailed description thereof will be omitted.

In the method for producing recombinant foot-and-mouth disease virus according to the present invention, the cells in the step (3) for injecting the recombinant plasmid constructed in the step (2) into the cells are obtained from the fetal tongue cells (ZZ-R) and the young hamster kidney cells BHK21) may be used.

In the method for producing recombinant foot-and-mouth disease virus according to the present invention, the recombinant virus is characterized in that a defensive antigen of the foot-and-mouth disease type SAT1 type WZ region is expressed.

In the method for producing recombinant foot-mouth virus according to the present invention, the recombinant virus is characterized by being a virus for foot-and-mouth disease.

The present invention also relates to a vaccine for the prevention of foot-and-mouth disease including the recombinant foot-and-mouth disease virus.

The vaccine of the present invention may be a vaccine live vaccine or an inactivated vaccine, and the vaccine may be manufactured using conventional vaccine production methods, so that detailed description thereof will be omitted.

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

Example 1 Production of Recombinant Plasmid pO-Manisa

The recombinant plasmid pO-Manisa was constructed by amplifying the entire O-Manisa virus gene (GenBank Accession No. AY593823.1) by PCR and inserting the O-Manisa gene into a plasmid (pBluescript SK II).

In the above, PCR amplification was carried out in the following manner.

Generation of cDNA for O-Manisa virus using random primers used in cDNA synthesis and specific primers capable of amplifying based on O-Manisa virus gene information (GenBank Accession No. AY593823.1 based on 5 'and 3' genes, respectively). The PCR conditions were as follows: 5X buffer (FINNZYMES, 10 μl), 10 mM dNTPs (1 μl), Phusion enzyme The reaction was carried out at 98 ° C for 30 seconds, followed by 25 cycles at 98 ° C for 10 seconds, at 65 ° C for 30 seconds, at 72 ° C for 2 minutes and 30 seconds, and finally at 72 ° C for 10 minutes in the capacity of sterilized distilled water (2U / Respectively.

1 shows a schematic diagram of a plasmid (pO-Manisa) into which a whole gene of a foot-and-mouth disease type O vaccine strain Manisa is inserted.

Example 2 Preparation of recombinant foot-and-mouth disease virus substituted with P1 site

Among the recombinant plasmids (pO-Manisa) prepared in Example 1, the gene coding for the P1 protein as a defense antigen was replaced with a gene coding for P1 protein (Gene Berkman Session Number AY593842), a defense antigen of foot-and-mouth type SAT1 type WZ locus (POm-SAT1-SA-P1) in which the recombinant gene was inserted into the plasmid having the nucleotide sequence of SEQ ID NO: 1.

The gene coding for the P1 protein was carried out as follows.

(GenBank Accession No. AY593842) using the forward primer (5'-GGAGCAGGTCAGTCGTCACCAGCT-3 ') of SEQ ID NO: 2 and the reverse primer (5'-CTGCTTGGCAGGTTTGACGAGGGC- After synthesizing the gene, it was amplified by PCR using this as a template.

The PCR conditions were 98 ° C for 30 seconds at 98 ° C and 10 ° C for 10 minutes at 98 ° C in 5X buffer (FINNZYMES, 10 μl), 10 mM dNTPs (1 μl), Phusion enzyme (2 U / μl, 0.5 μl) and sterilized distilled water Sec, 25 seconds at 65 占 폚 for 30 seconds, 72 占 폚 for 60 seconds, and final 72 占 폚 for 10 minutes.

A vector in which the P1 gene was deleted from the Omanisa gene was amplified by PCR using the forward primer (5'-CTTCTAAATTTGACCTGCTCAAATTGGCG-3 ') of SEQ ID NO: 4 and the reverse primer (5'-CTTGAGCCTTTTCTGGACCTTTGTTTTTCCA-3' .

At this time, the PCR conditions were 98 ° C for 30 seconds at 98 ° C and 10 seconds for 10 minutes at 98 ° C in 5X buffer (FINNZYMES, 10ul), 10mM dNTPs (1μl), Phusion enzyme (2U / , 25 seconds at 65 占 폚 for 30 seconds, 72 占 폚 for 2 minutes and 30 seconds, and finally 72 占 폚 for 10 minutes.

After that, a TAKARA Long Ligation kit was performed with the amplified P1 gene. Finally, it was confirmed that cloning was properly performed (pOm-SAT1-SA) through whole sequence analysis.

The recombinant foot-and-mouth disease virus was recovered by reacting the obtained recombinant plasmid (pO1m-SAT1-SA) with a restriction enzyme, securing the virus in BHKT7-9 cells (cell line expressing T7 RNA polymerase) Fetal tongue) cells and BHK21 (young hamster kidney) cells, and the cytopathic effect was confirmed in BHK21 cells and IBRS-2 (pig kidney) cells.

FIG. 2 is a genomic diagram of a recombinant FMD virus expressing a defensive antigen of the foot-and-mouth type SAT 1 type WZ region. In the whole genome of the foot-and-mouth disease virus O type Manisa, / 5sa / 61).

Fig. 3 is a schematic diagram of a plasmid into which the defective protein gene of the foot-and-mouth disease foot-and-mouth disease type SAT1 WZ region is inserted.

FIG. 4 is a photograph showing a plaque formed in a small intestine cell (LF-BK) of a virus recovered from a plasmid in which a defensive protein gene of foot-and-mouth disease type SAT1 type WZ region is inserted.

FIG. 5 shows that a protein expressed in recombinant foot-and-mouth disease virus according to the present invention shows a foot-and-mouth antigen positive reaction using a liver antigen kit. 25 μl of the recovered virus culture supernatant can be confirmed with a positive antigen kit (Svanova Co. Ltd) for a positive response to the structural protein (SP) against foot-and-mouth disease.

6 shows that recombinant foot-and-mouth disease virus (BFR) according to the present invention inhibits the expression of recombinant FMDVs in young hamster kidney cells (BHK21), black goat kidney cells (BGK), porcine kidney cells (IBRS- -BK) to form virus titer.

Although the present invention has been described and illustrated in detail, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the scope of the present invention as defined by the appended claims. It will be understood that various modifications and changes may be made in the present invention.

Foot and Mouth Disease By providing a foot-and-mouth disease vaccine using the recombinant foot-and-mouth disease virus according to the present invention having the ability of expressing the SAT 1 type WZ locus type protective antigen, it can contribute to the treatment and / or prevention of foot-and-mouth disease.

<110> Animal, Plant and Fisheries Quarantine and Inspection Agency <120> Foot-and-mouth disease virus expressing P1-protective antigen          SAT1-WZ topotype and the manufacturing methods <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 11127 <212> DNA <213> Artificial Sequence <220> <223> pOm-SAT1-SA-P1 <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 aggtcagtcg tcaccagcta cagggacaca aaatcaatct 2400 ggtaacactg gaagcatcat caacaactac tacatgcagc agtaccagaa ctcaatggac 2460 acccaacttg gcgacaacgc catttcaggt ggttccaatg aggggtcgac cgacaccacg 2520 tcgacccaca ccaacaacac tcaaaacaat gactggttct ccaaattggc acaatcggct 2580 ttctccgggc ttgttggcgc gcttctggcc gacaagaaga ccgaggagac cactctgctt 2640 gaggaccgta tcatgactac cagtcacggt accaccacat caaccacaca gagctcggtg 2700 ggcgtcacct acgggtatgc tctggccgat aagttcctcc ctggcccaaa caccaacggg 2760 ctggagacga gagtggaaca agcagagagg ttcttcaaac acaagctttt tgattggaca 2820 cttgaacaga aatttggcac aacctacgtt ctggaacttc ccacagatca taagggtatc 2880 tacggacaac tggttgactc acatgcgtac atccgcaacg gatgggacgt ccaggtctca 2940 gccactgcca cccagttcaa cggaggctgc ctgttggtag ccatggtgcc tgagctttgc 3000 aaattggccg acagggagaa gtaccaactt actcttttcc cacaccagtt cctgaaccca 3060 cgcaccaaca ccacagcaca catccaggta ccgtacttgg gtgttgacag acacgaccag 3120 gggactcgcc acaaggcgtg gactctcgtc gtgatggtgc tggcaccata caccaacgac 3180 cagaccattg gatcaacaaa agctgaggtc tacgtgaaca ttgcaccaac caacgtttac 3240 gtggccggtg agaaacccgc caagcaaggg attctccccg tggccgtttc cgacggttac 3300 ggtggcttcc aaaacactga ccccaagaca tcggacccca tttacgggca cgtgtacaac 3360 ccggcacgca cgctctaccc cggcaggttc accaacctgt tggacgtagc agaagcctgc 3420 cccacactgc ttgatttcaa cggggtccca tatgtccaaa cccagaacaa ctctggatca 3480 aaagttctca cacgtttcga tttggctttt gggcataaaa ccatgaagaa tacatacatg 3540 tctggtctgg cccagtactt cgcacagtac agtggcactc tcaatctgca tttcatgtac 3600 actggcccca ccaacaacaa ggccaagtac atggtggcgt acatcccacc tggcacacac 3660 cctctccccg acacaccgga gatggcgtca cactgctacc acgctgagtg ggatactgga 3720 ctgaactcaa cattcacctt caccgtgccg tacatttcgg cagcggacta cgcctacacc 3780 tacgctgacg agcccgaaca ggcttcagtg caaggttggg ttggtgtgta tcagatcact 3840 gacacacatg aaaaggacgg agccgtcatc gtcaccgtga gtgcaggccc cgactttgag 3900 ttcaggatgc ccatcagtcc atcgcgccag acaacctctg caggtgaagg cgcggaccca 3960 gtcaccacag acacgtccga acacggaggt gcctccagag tcgcccgtcg ggcccacacc 4020 gacgtggcgt tccttcttga ccggttcact ctggtcggga agaccaagaa caaccaattg 4080 gttctggacc tcttggacac caaagagaaa tcactggtcg gcgcactcct gcgcgcggcc 4140 acgtactact tctctgactt ggaagtggca tgtgttggga ccaatacatg ggtgggctgg 4200 acgcccaatg gtagtccagt gatgagcgaa gtgggcgaca acccagtcgt cttctcgcac 4260 aacggcacca ctcgttttgc actcccttac actgcacccc accgggtgct tgccacagtc 4320 tacaatggtg actgcaagta caaacccact ggcaccgcac cccgcgaaaa catccgcggt 4380 gacctcgcaa ctcttgctgc gcggattgct agtgagaccc acatcccgac gacatttaac 4440 tacgggatga tttacacaca ggcagaggtg gacgtgtacc tgaggatgaa gagagcagaa 4500 ctctactgcc ctcgacccgt tctcacgcac tacgaccacg gcaacaagga tcgtcacaag 4560 acggccctcg tcaaacctgc caagcagctt ctaaattttg acctgctcaa attggcggga 4620 gatgtggagt ccaaccctgg gcccttcttc ttctccgacg tcaggtcaaa tttctcaaaa 4680 ctggtagaaa ccatcaatca gatgcaggag gacatgtcaa caaaacacgg gcctgacttt 4740 aaccggttgg tgtccgcatt tgaggaattg gccactggag tgaaggctat cagggccggt 4800 ctcgacgagg ccaaaccctg gtacaaactc atcaagctcc tgagccgctt gtcgtgcatg 4860 gccgctgtag cagcacggtc aaaggaccca gtccttgtgg ccatcatgct ggctgacacc 4920 ggtcttgaga ttctggacag cacctttgtc gtgaagaaga tctccgactc gctctccagt 4980 ctctttcacg tgccggcccc cgtcttcagt ttcggagccc cgattctgtt ggccgggttg 5040 gtcaaagtcg cctcgagttt cttccggtcc acacccgaag accttgagag agcagaaaaa 5100 cagctcaaag cacgtgacat taacgacata ttcgccattc tcaagaacgg cgagtggctg 5160 gtcaagctga tccttgccat ccgcgactgg atcaaagcgt ggatcgcctc agaagaaaag 5220 tttgtcacca tgacggactt ggtgcctggt atccttgaaa agcagcggga tctcaacgac 5280 ccgagtaagt acaaggaagc caaggagtgg ctcgacaacg cgcgccaggc gtgtttgaag 5340 agcgggaacg ttcacattgc caatttgtgc aaagtggtcg ccccggcacc cagcaagtcg 5400 agacccgaac ccgtggtcgt ttgcctccgc ggcaaatccg gccaggggaa gagtttcctt 5460 gcgaacgtgc tcgcgcaagc aatctccacc cacttcaccg gcagaactga ttcggtttgg 5520 tactgcccgc ctgaccctga ccacttcgac ggttacaacc agcagaccgt tgtcgtgatg 5580 gacgatttgg gccagaaccc cgatggcaag gacttcaagt acttcgccca gatggtttcg 5640 accacggggt tcatcccgcc catggcctcg cttgaggaca aaggcaagcc tttcaacagc 5700 aaagtcatca ttgctaccac caacctgtac tcgggtttca ccccgagaac aatggtgtgt 5760 cctgacgcgc tgaaccggag gttccacttt gacatcgacg tgagtgccaa ggacgggtac 5820 aaagttaaca acaaattgga cataatcaaa gctcttgaag acacccacac caacccagtg 5880 gcgatgttcc aatacgactg tgcccttcta aacggtatgg cagttgaaat gaagagaatg 5940 caacaggata tgttcaagcc tcaaccaccc ctccagaacg tgtaccaact cgttcacgag 6000 gtgattgaac gggtcgagct ccacgagaag gtgtcgagcc acccgatttt caaacagata 6060 tcaattcctt cccaaaagtc tgtgttgtac ttcctcattg agaaaggcca acacgaagca 6120 gcaattgaat tctttgaggg aatggtgcat gactccatca aggaagagct ccggcccctc 6180 atccaacaga cctcatttgt gaaacgcgct tttaagcgcc tgaaggaaaa ctttgagact 6240 gttgccctgt gtttgactct tttggcaaac atagtgatca tgatccgcga gactcgcaag 6300 agacaacaga tggtggacga tgcagtgaat gactacattg agaaggcaaa catcaccaca 6360 gatgacaaga ctcttgacga ggcggaaaag aaccctctag agaccagcgg tgccagcact 6420 attggtttca gagagagaac tctcccgggg cacaaggcga gcgatgacgt gagctccgag 6480 cccgccaaac ccgtggagga ccgaccacaa gctgaagggc cctacgccgg accacttgag 6540 cgtcagaaac ctctgagagt gaaaaccaag ttgccacaac aggagggacc ctacgctggc 6600 ccgatggata gacagaaacc gttgaaagtg agagcaagag ccccggtcgt gaaggaggga 6660 ccctacgagg gaccggtgaa gaagcctgtc gctttgaaag tgaaagccaa gaacttgatt 6720 gtcactgaga gtggtgcccc accgaccgac ttgcagaaga tggtcatggg caacactaag 6780 cctgttgagc tcatcctcga cgggaagacg gtagccatct gctgtgctac cggagtgttt 6840 ggcactgcct acctcgtacc tcgtcacctc ttcgcggaga agtacgacaa gataatgttg 6900 gacggtagag ccatgacaga cagtgactac agagtgtttg agtttgagat taaagtaaaa 6960 ggacaggaca tgctctcaga cgctgcactc atggtgcttc accgtgggaa ccgcgtgaga 7020 gacatcacga aacattttcg tgacacagca agaatgaaga aaggcacccc cgttgtcggt 7080 gtgatcaaca acgccgacgt tgggagactg attttctctg gagaggccct tacctacaaa 7140 gacattgtag tgtgcatgga tggagacacc atgccgggcc tgtttgccta cagagccgcc 7200 accaaggctg gttactgcgg gggagccgtt ctcgccaagg acggagccga cacattcatc 7260 gtggcaccc actccgcagg tggtaacgga gttggatact gctcgtgcgt gtccaggtcc 7320 atgctcctga aaatgaaggc acacattgac cctgaaccac accacgaggg gttgattgtt 7380 gataccagag atgtggaaga gcgcgtgcat gtcatgcgta aaaccaagct tgcacccacc 7440 gtggcacacg gtgtgtttaa ccctgaattt ggtcccgctg ccttgtccaa caaggacccg 7500 cggctgaacg aaggggttgt cctcgatgaa gtcatcttct ccaaacacaa gggagacacg 7560 aaaatgtctg aggaggacaa agcgctgttc cgccgctgcg ctgccgacta cgcgtcgcac 7620 ttgcacagcg tgctggggac ggcaaatgcc ccattgagca tctatgaggc catcaaaggc 7680 gtcgacgggc tcgatgccat ggagccggac accgcgcccg gcctcccctg ggccctccag 7740 gggaaacgcc gtggtgcgtt gattgacttc gagaacggca cggtcggacc cgaagtcgag 7800 gctgccctaa agctcatgga gaaaagagag tacaaatttg cttgccagac cttcctgaaa 7860 gacgagattc gtccgatgga aaaagtacgt gctggcaaga ctcgcattgt cgacgttttg 7920 cccgtggaac acattcttta caccaggatg atgattggca gattctgtgc tcaaatgcac 7980 acaaacaatg gaccgcagat tggctcagcg gtcggttgca atcctgatgt tgattggcaa 8040 agatttggca cacattttgc tcagtacaga aacgtgtggg atgtggacta ttcggccttt 8100 gatgctaacc actgcagtga cgcaatgaac atcatgtttg aggaggtatt tcgcacagac 8160 ttcggtttcc acccaaatgc tgagtggatt ctgaagactc ttgtgaacac ggagcacgcc 8220 tatgagaaca aacgtatcac tgttgagggc gggatgccgt ctggctgttc cgcgacaagc 8280 atcatcaaca caattttgaa caacatttat gtgctctacg ctcttcgtag acactatgag 8340 ggagttgagc tggacaccta caccatgatc tcctacggag atgacatcgt ggttgcaagt 8400 gactacgatc tggattttga ggctctcaaa ccccacttca aatctcttgg tcaaaccatc 8460 actccagctg acaaaagcga caaaggtttt gttcttggtc actccattac cgatgtcact 8520 ttcctcaaaa gacacttcca catggactat ggaactgggt tttacaaacc tgtgatggcc 8580 tcaaagaccc tcgaggccat tctctccttt gcacgccgtg ggaccataca ggagaagttg 8640 atctccgtgg caggactcgc cgtccactca ggacctgacg agtaccggcg tctctttgag 8700 cccttccagg gtctcttcga gattccaagc tacagatcac tttacctgcg ttgggtgaac 8760 gccgtgtgcg gtgacgcata atccctcaga tgtcacaatt ggcagaaaga ctctgaggcg 8820 agcgacgccg taggagtgaa aagcccgaaa gggcttttcc cgcttcctat tccaaaaaaa 8880 aaaaaaaaaa actagttcta gagcggccgc caccgcggtg gagctccagc ttttgttccc 8940 tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa 9000 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct 9060 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc 9120 agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg 9180 gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 9240 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag 9300 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa 9360 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc 9420 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc 9480 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 9540 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt 9600 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc 9660 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc 9720 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag 9780 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg 9840 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa 9900 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag 9960 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact 10020 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa 10080 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt 10140 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag 10200 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca 10260 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc 10320 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt 10380 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg 10440 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca 10500 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg 10560 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca 10620 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg 10680 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct 10740 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca 10800 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca 10860 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg 10920 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac 10980 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt 11040 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc 11100 cgcgcacatt tccccgaaaa gtgccac 11127 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 ggagcaggtc agtcgtcacc agct 24 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 ctgcttggca ggtttgacga gggc 24 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 cttctaaatt ttgacctgct caaattggcg 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 cttgagcctt ttctggacct ttgttttcca 30

Claims (9)

Among the whole gene regions of the foot-and-mouth disease O-type vaccine strain Manisa, the gene coding for the P1 protein composed of VP1, VP2, VP3 and VP4 as protective antigens of the foot-and-mouth disease type O vaccine Manisa was selected as a foot-and-mouth disease strain having the nucleotide sequence of Gene Berkman Session Number AY593842 A recombinant foot-and-mouth disease virus comprising a plasmid having the nucleotide sequence of SEQ ID NO: 1, wherein the plasmid is substituted with a gene encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as a SAT1 WZ type localization antigen. delete 2. The recombinant FMD virus according to claim 1, wherein the recombinant virus expresses the defensive antigen of the foot-and-mouth disease type SAT1 type WZ region. The recombinant foot-and-mouth disease virus according to claim 1 or 3, wherein the recombinant virus is a foot-and-mouth disease vaccine virus. (One). Inserting a whole gene of foot-and-mouth disease type O vaccine strain Manisa into a plasmid;
(2). Among the entire genes of the foot-and-mouth disease O-type vaccine strain Manisa of the plasmid obtained in the above step (1), a gene encoding a P1 protein composed of VP1, VP2, VP3 and VP4 as protective antigens of the foot- A recombinant plasmid having a recombinant gene inserted therein was constructed by substituting a gene coding for P1 protein composed of VP1, VP2, VP3 and VP4 as a foot-and-mouth type SAT1 WZ-type protective antigen having a nucleotide sequence of the number AY593842, Having the nucleotide sequence of SEQ ID NO: 1; And
(3). And introducing the recombinant plasmid constructed in the step (2) into a cell to proliferate the recombinant FMD virus. [6] The method according to claim 5, wherein the cell is at least one selected from the group consisting of fetal goat fetus tongue (ZZ-R) and young hamster kidney (BHK21) cells. 6. The method according to claim 5, wherein the recombinant virus expresses the defensive antigen of the foot-and-mouth type SAT1 type WZ region. The method for producing recombinant foot-and-mouth disease virus according to any one of claims 5 to 7, wherein the recombinant virus is a foot-and-mouth disease vaccine virus. A vaccine for the prevention of foot-and-mouth disease comprising the recombinant foot-and-mouth disease virus of claim 1 or 3.

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