KR101629345B1 - Foot and mouth disease virus expressing P1-protective antigen of Asia1 type, IV genotype and the manufacturing method - 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, and a gene encoding a P1 protein which is a protective antigen of foot-and-mouth disease type 1 serotype type IV virus The present invention relates to a recombinant foot-and-mouth disease virus comprising a plasmid in which a recombinant gene substituted with a substituted plasmid is inserted, and a method for producing the same.

Description

FIELD OF THE INVENTION The present invention relates to recombinant foot-and-mouth disease virus (hereinafter referred to as " foot-and-mouth disease virus "

The present invention relates to a recombinant foot-and-mouth disease virus which expresses a defensive antigen of foot-and-mouth disease type 1 serotype IV virus and a method for producing the same. More particularly, the present invention relates to a recombinant foot- A recombinant foot-and-mouth disease virus comprising a plasmid in which a gene coding for a P1 protein as a protective antigen is inserted with a recombinant gene substituted with a gene coding for P1 protein which is a protective antigen of foot-and-mouth disease type 1 serotype IV virus and a preparation method thereof will be.

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) (Korean Patent Laid-Open No. 10-2011-0038380).

Among the O-type foot-and-mouth disease viruses, there are more than 8 regional types (Korean Veterinary Journal, Vol. 48, No. 12, pp. 702-724, 2012). 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 has been internationally contraindicated to be used as an attenuated vaccine, which has so far been reduced in pathogenicity to foot-and-mouth disease virus, since the 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.

Foot-and-mouth disease virus type O Manisa is known to be the most widely used vaccine virus in O-type vaccines, and it is possible to defend against viruses in ME-SA (Middle East-South Asia, including the Middle East-South Asia type and PanAsia type) .

However, in addition to the O type, there are also Asian type 1 and A type in Korea, and the Asian type 1 serotype can be classified into the group I-VI. The above-mentioned serotype 1 of Asia is confined to the Asian region, but the virus has been transmitted to Turkey and Greece in 2000, and an increase in the serotype 1 of Asian serotype has recently been reported (Korean J. Vet. Serv. 32 (4): 315-323 (2009)).

Recently, it has occurred in East Asia and has been complicated. It occurred in Southeast Asia such as Afghanistan, India, Iran, Malaysia, Nepal, Pakistan and Thailand. From the end of 2004 to 2006, , Mongolia, East Russia, Tajikistan, and Vietnam (Korean J. Vet. Serv. 32 (4): 315-323 (2009)).

The Asian 1 serotype genotype IV is a genotype that continuously develops in Southeast Asia and is propagated to China, which is known to be a major propagation pathway in Asia, and genotypes IV, V and VI are generated around the country including this genotype. (Korean J. Vet. Serv. 32 (4): 315-323 (2009)), and the development of a vaccine against these viruses is urgently required.

In order to adequately defend against the foot-and-mouth disease type 1 serotype IV virus, the inventors of the present invention have found that, based on a viral vector obtained by cloning the whole genome of foot-and-mouth disease virus O manisa vaccine into a plasmid, A new recombinant foot-and-mouth disease virus that expresses a defense protein that can defend against the Asian 1 serotype IV virus by replacing the structural protein with the structural protein gene of the Asian 1 serotype IV virus (Asia1 / VN / LC04 / 2005) The present invention has been completed.

Therefore, the object of the present invention is to provide a method for preventing and treating the above-mentioned Asia1 / VN / LC04 / 2005 deficiency by inserting P1 gene of foot-and-mouth disease type 1 serotype IV virus (Asia1 / VN / LC04 / 2005) And to provide a recombinant foot-and-mouth disease virus which can be usefully used as a vaccine seedling.

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 1 serotype IV virus which is prevalent in Korea,

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, which is substituted with a gene encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as a protective antigen of foot-and-mouth disease asian serotype 1 genotype IV virus having the nucleotide sequence of GenBank accession number GU125646 To provide a recombinant foot-and-mouth virus comprising the inserted plasmid.

The present invention also provides a method for producing a recombinant human O-type vaccine, comprising the steps of: (1) inserting a whole gene of a foot-and-mouth type O-type vaccine strain Manisa into a plasmid; (2) VP3 and VP4 was transfected with a plasmid encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as a protective antigen of foot-and-mouth disease type 1 serotype type IV virus having the nucleotide sequence of Gene Berkman session number GU125646 And (3) injecting the recombinant plasmid constructed in the step (2) into a cell to proliferate the recombinant foot-and-mouth disease virus And a manufacturing method thereof.

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 treats part of the foot-and-mouth disease virus O Manisa gene in a laboratory such as disinfectant experiment or antivirus experiment by weakening the pathogenicity to an animal.

In addition, the recombinant foot-and-mouth disease virus according to the present invention can effectively prevent the viral localization occurring in Korea, East Asia, China, Taiwan, etc., while the virus titer does not greatly differ from 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.
Fig. 2 shows a genomic diagram of a recombinant FMDV virus expressing a protective antigen of foot-and-mouth disease type 1 serotype IV virus.
FIG. 3 is a schematic diagram of a recombinant plasmid (pO1m-AsVNLC04-P1) into which a defensive protein gene of foot-and-mouth disease type 1 serotype IV virus is inserted.
FIG. 4 shows that a protein expressed in recombinant foot-and-mouth disease virus according to the present invention shows a FMF-positive reaction using a liver antigen kit.
5 is a photograph showing virus-infected cells (detection of fluorescent antibody method) and cytopathic effect in foot-and-mouth disease virus infected cells.
FIG. 6 is a graph showing that recombinant foot-and-mouth disease virus according to the present invention grows in a hamster cell line (BHK-21) to form a 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 protective antigen of foot-and-mouth disease type 1 serotype IV virus having a nucleotide sequence of SEQ ID NO: It's about viruses.

In the recombinant FMD virus of the present invention, the plasmid into which the recombinant gene is inserted is characterized by having 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 in that a defensive antigen of the foot-and-mouth disease type 1 serotype type IV virus is expressed.

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

The present invention also provides a method for producing a recombinant human O-type vaccine, comprising the steps of: (1) inserting a whole gene of a foot-and-mouth type O-type vaccine strain Manisa into a plasmid; (2) VP3 and VP4 was transfected with a plasmid encoding a P1 protein consisting of VP1, VP2, VP3 and VP4 as a protective antigen of foot-and-mouth disease type 1 serotype type IV virus having the nucleotide sequence of Gene Berkman session number GU125646 And (3) injecting the recombinant plasmid constructed in the step (2) into a cell to proliferate the recombinant foot-and-mouth disease virus And a manufacturing method thereof.

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 a recombinant foot-and-mouth virus according to the present invention, the recombinant virus is characterized in that a protective antigen of the foot-and-mouth disease type 1 serotype type IV virus is expressed.

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 The PCR conditions were as follows: 5X buffer (FINNZYMES, 10 μl), 10 mM dNTPs (1 μl), Phusion enzyme (2 U / μl, And 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 volume of sterilized distilled water (35.5 μl).

In addition, the 3B region of some genes (22-36 bp in the 3B region, gagcgtcagaaacct) and the deletion (3B region in the 3B region) were amplified using the commonly used in vitro mutagenesis method of the entire gene (GenBank Accession No. AY593823.1) (37-105 bps region, ctgagagtga aaaccaagtt gccacaacag gagggaccct acgctggccc gatggataga cagaaaccg) was prepared. The recombinant plasmid pO-Manisa-3B2d was constructed.

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-3B2d) prepared in Example 1, the gene coding for the P1 protein as a defense antigen was transferred to the Asia1 / VN / LC04 / 2005 virus (GenBank Accession No. (PO1m-AsVNLCO4-P1) having the recombinant gene inserted therein was prepared by substituting the gene coding for the P1 protein, which is a protective antigen of the recombinant virus,

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

VN / LC04 / 2005 virus (GenBank Accession No. GU125646) using the forward primer (5'-GGAGCTGGGCAATCCAGCCCGGCGAC-3 ') of SEQ ID NO: 2 and the reverse primer (5'-CTGCTTCTCGGGCGCAATGATTTCCTG- Were amplified by PCR.

The PCR conditions were 98 ° C for 30 seconds at 98 ° C for 10 seconds, 5 minutes at 98 ° C, 5 minutes in the presence of 5 × buffer (FINNZYMES, 10 μl), 10 mM dNTPs (1 μl), Phusion enzyme (2 μl, 0.5 μl), and sterilized distilled water The reaction was carried out at 65 ° C for 30 seconds, 72 ° C for 60 seconds, 25 cycles, and finally at 72 ° C for 10 minutes.

A vector in which the P1 gene was removed from the Omanisa gene was amplified by PCR using the forward primer (5'-CTTCTAAATTTTACCTGCTCAAATTGACCTGCTCAAATTGGCGGG-3 ') of SEQ ID NO: 4 and the reverse primer (5'-CTTGAGCCTTTTCTGGACCTTTTTTTTCCAC-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 cycles at 65 占 폚 for 30 seconds, 72 占 폚 for 2 minutes and 30 seconds, and final 72 占 폚 for 10 minutes.

Thereafter, the amplification reaction with the P1 gene (TAKARA Long Ligation Kit) was performed, and finally cloning (pO1m-AsVNLC04-P1) was confirmed through the whole base sequence analysis.

After recovery of the recombinant FMD virus, the recombinant plasmid (pO1m-AsVNLC04-P1) was reacted with restriction enzymes, and the virus was obtained from BHKT7-9 cells (cell line expressing T7 RNA polymerase) (Fetal tongue) and BHK-21 (young hamster kidney) cells, and the cytopathic effect was confirmed in BHK-21 cells.

FIG. 2 is a genomic diagram of a recombinant FMDV virus expressing a protective antigen of foot-and-mouth disease type 1 serotype IV virus. PO1m-AsVNLC04-P1 is a genome of a foot-and- Site was replaced with the P1 region of foot-and-mouth disease Asia 1 serotype genotype IV virus (Asia1 / VN / LC04 / 2005).

Fig. 3 is a schematic diagram of a plasmid into which a defective protein gene of foot-and-mouth disease type 1 serotype IV virus is inserted.

FIG. 3 is a schematic diagram of a recombinant plasmid (pO1m-AsVNLC04-P1) into which a defensive protein gene of foot-and-mouth disease type 1 serotype IV virus is inserted.

FIG. 4 shows that a protein expressed in recombinant foot-and-mouth disease virus according to the present invention shows a FMF-positive reaction using a liver antigen kit. 25 μl of the recovered viral culture supernatant can be confirmed with a positive antigen kit (PBM Co. Ltd.) for a positive response to the structural protein (SP) against foot-and-mouth disease.

5 is a photograph showing virus-infected cells (detection of fluorescent antibody method) and cytopathic effect in foot-and-mouth disease virus infected cells. Recovered viruses in the laboratory were infected with the cells, so that they could detect virus infection by fluorescence antibody method and showed the effect of denaturing the cells.

FIG. 6 is a graph showing that recombinant foot-and-mouth disease virus according to the present invention grows in a hamster cell line (BHK-21) to form a virus titer. Referring to FIG. 6, the recombinant foot-and-mouth disease virus of the present invention shows a virus titer similar to that of an outdoor virus.

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.

By providing a foot-and-mouth disease vaccine using the recombinant foot-and-mouth disease virus according to the present invention, which possesses the protective antigen-expressing ability of foot-and-mouth disease Asian genotype 1 genotype IV virus, 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 expression P1-protective antigen of          Asia1 type, IV genotype and the manufacturing method <160> 5 <170> Kopatentin 1.71 <210> 1 <211> 11019 <212> DNA <213> Artificial Sequence <220> <223> pO1m-AsVNLC04-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 tgggcaatcc agcccggcga ccgggtcgca aaatcaatca 2400 ggcaacactg gaagcatcat taacaattac tacatgcagc agtaccagaa ctccatggac 2460 acgcaacttg gagacaacgc tatcagcgga ggctccaacg agggttccac ggacaccacg 2520 tccacacaca caaacaacac ccaaaacaat gattggttct cacgcttggc cagctcggcc 2580 tttagcggac tgtttggtgc tcttttggct gacaagaaaa cggaggagac aactctgctt 2640 gaagaccgca ttctcaccac cagaaacggc cacacgacgt cgacgacaca gtcgagcgtc 2700 ggcgttacct acggttacgc catggccgaa gacgcggtgt cagggcccaa cacctcgggc 2760 ctagagactc gtgtgttaca agctgagcgg ttcttcaaga aacacctgtt tgattggaca 2820 ccgaatttgg aatttgggca ctgtcattac ctggaactcc cctctgaaca caaaggcgtc 2880 tatggaagcc tccagaactc ttatgcttac atgaggaacg ggtgggacat tgaggttacc 2940 gctgttggaa accaattcaa cggtggttgt cttcttgtcg cgcttgtgcc agaattgaaa 3000 acccttggaa cacggcaaaa gtaccaactg actcttttcc cccaccagtt tgtaaaccca 3060 cgcaccaaca tgacggctca catcaacgtg ccgtacgtgg gtgttaacag gtatgatcaa 3120 tacgcgctcc acaaaccatg gacgctcgta gtgatggtgg tggcgccact cactgtcaaa 3180 actggtggat ctgaacaaat caaggtttac atgaatgcag caccgaccta cgtgcacgtg 3240 gcgggagagc taccctcgaa ggagggaata gtccccgttg cgtgtgcgga tggatatggc 3300 aacatggtga ccacggaccc gaagacggct gaccccgttt atgggaaagt gttcaacccc 3360 ccccggacga acctgcccgg gcggttcacg aacttccttg atgttgcgga ggcctgtcca 3420 accttcctcc gcttcggaga agttccgttt gtgaagacgg tgaacaccgg tgaccgtttg 3480 cttgccaagt ttgacgtgtc cctctctgca gggcacatgg ctaacaccta cttggctggc 3540 ttggcgcagt actacacaca gtacagcggc accatgaacg ttcacttcat gtttaccggc 3600 cccacagatg ccaaagcccg ctatatggtg gcttacgtgc ccccgggcat ggaaccaccc 3660 actgaccctg agcgggctgc acactgcatt cattctgagt gggacaccgg actcaactcc 3720 aaattcactt tctccatacc atacctttct gctgctgact acgggtacac tgcttccaac 3780 gtggccgaga ccacaagtgt gcagggctgg gtgtgcatct accagatcac acatggcaaa 3840 gctgagggtg acgccttggt cgtttccgtc agcgccggca aggactttga gtttcgactg 3900 ccagtcgatg ctcgccagca aaccacaacc acaggcgagt cagcagaccc cgtcaccact 3960 acggtggaaa actacggggg agtgacgcag gcggcccgac gtctccacac cgacgttgcc 4020 ttcgttctcg acaggtttgt gaaacttacc cagcccaaga gcactcaggt tcttgacctc 4080 atgcagatac cctcacacac actggttgga gccctgcttc ggtctgcgac gtactacttc 4140 tcagacctgg agattgcgct tgtccacaca ggcccggtca cgtgggtgcc caacggcgca 4200 cctaaggttg ccctggacaa ccagaccaat cccactgcct accaaaagca gcccattacc 4260 cgtctggcgc tcccttacac cgccccccac cgtgtgctgg caacagtgta caacgggaag 4320 acggcatacg gggagacgac cacgaggcgt ggtgatcttg ctgccctagc acaaagggtg 4380 agcaggcagt tgccaacctc cttcaactac ggcgcagtga aggccgaaag catcacagag 4440 ttgttggtcc gcatcaaacg cgcggagaca tactgcccca ggcctctgtt ggctcttgac 4500 accgtccaag accgccgcaa acaggaaatc attgcgcccg agaagcagct tctaaatttt 4560 gacctgctca aattggcggg agatgtggag tccaaccctg ggcccttctt cttctccgac 4620 gtcaggtcaa atttctcaaa actggtagaa accatcaatc agatgcagga ggacatgtca 4680 acaaaacacg ggcctgactt taaccggttg gtgtccgcat ttgaggaatt ggccactgga 4740 gtgaaggcta tcagggccgg tctcgacgag gccaaaccct ggtacaaact catcaagctc 4800 ctgagccgct tgtcgtgcat ggccgctgta gcagcacggt caaaggaccc agtccttgtg 4860 gccatcatgc tggctgacac cggtcttgag attctggaca gcacctttgt cgtgaagaag 4920 atctccgact cgctctccag tctctttcac gtgccggccc ccgtcttcag tttcggagcc 4980 ccgattctgt tggccgggtt ggtcaaagtc gcctcgagtt tcttccggtc cacacccgaa 5040 gaccttgaga gagcagaaaa acagctcaaa gcacgtgaca ttaacgacat attcgccatt 5100 ctcaagaacg gcgagtggct ggtcaagctg atccttgcca tccgcgactg gatcaaagcg 5160 tggatcgcct cagaagaaaa gtttgtcacc atgacggact tggtgcctgg tatccttgaa 5220 aagcagcggg atctcaacga cccgagtaag tacaaggaag ccaaggagtg gctcgacaac 5280 gcgcgccagg cgtgtttgaa gagcgggaac gttcacattg ccaatttgtg caaagtggtc 5340 gccccggcac ccagcaagtc gagacccgaa cccgtggtcg tttgcctccg cggcaaatcc 5400 ggccagggga agagtttcct tgcgaacgtg ctcgcgcaag caatctccac ccacttcacc 5460 ggcagaactg attcggtttg gtactgcccg cctgaccctg accacttcga cggttacaac 5520 cagcagaccg ttgtcgtgat ggacgatttg ggccagaacc ccgatggcaa ggacttcaag 5580 tacttcgccc agatggtttc gaccacgggg ttcatcccgc ccatggcctc gcttgaggac 5640 aaaggcaagc ctttcaacag caaagtcatc attgctacca ccaacctgta ctcgggtttc 5700 accccgagaa caatggtgtg tcctgacgcg ctgaaccgga ggttccactt tgacatcgac 5760 gtgagtgcca aggacgggta caaagttaac aacaaattgg acataatcaa agctcttgaa 5820 gacacccaca ccaacccagt ggcgatgttc caatacgact gtgcccttct aaacggtatg 5880 gcagttgaaa tgaagagaat gcaacaggat atgttcaagc ctcaaccacc cctccagaac 5940 gtgtaccaac tcgttcacga ggtgattgaa cgggtcgagc tccacgagaa ggtgtcgagc 6000 cacccgattt tcaaacagat atcaattcct tcccaaaagt ctgtgttgta cttcctcatt 6060 gagaaaggcc aacacgaagc agcaattgaa ttctttgagg gaatggtgca tgactccatc 6120 aaggaagagc tccggcccct catccaacag acctcatttg tgaaacgcgc ttttaagcgc 6180 ctgaaggaaa actttgagac tgttgccctg tgtttgactc ttttggcaaa catagtgatc 6240 atgatccgcg agactcgcaa gagacaacag atggtggacg atgcagtgaa tgactacatt 6300 gagaaggcaa acatcaccac agatgacaag actcttgacg aggcggaaaa gaaccctcta 6360 gagaccagcg gtgccagcac tattggtttc agagagagaa ctctcccggg gcacaaggcg 6420 agcgatgacg tgagctccga gcccgccaaa cccgtggagg accgaccaca agctgaaggg 6480 ccctacgccg gaccacttaa gaaaccagtg gcattgaaag tgagagcaag agccccggtc 6540 gtgaaggagg gaccctacga gggaccggtg aagaagcctg tcgctttgaa agtgaaagcc 6600 aagaacttga ttgtcactga gagtggtgcc ccaccgaccg acttgcagaa gatggtcatg 6660 ggcaacacta agcctgttga gctcatcctc gacgggaaga cggtagccat ctgctgtgct 6720 accggagtgt ttggcactgc ctacctcgta cctcgtcacc tcttcgcgga gaagtacgac 6780 aagataatgt tggacggtag agccatgaca gacagtgact acagagtgtt tgagtttgag 6840 attaaagtaa aaggacagga catgctctca gacgctgcac tcatggtgct tcaccgtggg 6900 aaccgcgtga gagacatcac gaaacatttt cgtgacacag caagaatgaa gaaaggcacc 6960 cccgttgtcg gtgtgatcaa caacgccgac gttgggagac tgattttctc tggagaggcc 7020 cttacctaca aagacattgt agtgtgcatg gatggagaca ccatgccggg cctgtttgcc 7080 tacagagccg ccaccaaggc tggttactgc gggggagccg ttctcgccaa ggacggagcc 7140 gacacattca tcgttggcac ccactccgca ggtggtaacg gagttggata ctgctcgtgc 7200 gtgtccaggt ccatgctcct gaaaatgaag gcacacattg accctgaacc acaccacgag 7260 gggttgattg ttgataccag agatgtggaa gagcgcgtgc atgtcatgcg taaaaccaag 7320 cttgcaccca ccgtggcaca cggtgtgttt aaccctgaat ttggtcccgc tgccttgtcc 7380 aacaaggacc cgcggctgaa cgaaggggtt gtcctcgatg aagtcatctt ctccaaacac 7440 aagggagaca cgaaaatgtc tgaggaggac aaagcgctgt tccgccgctg cgctgccgac 7500 tacgcgtcgc acttgcacag cgtgctgggg acggcaaatg ccccattgag catctatgag 7560 gccatcaaag gcgtcgacgg gctcgatgcc atggagccgg acaccgcgcc cggcctcccc 7620 tgggccctcc aggggaaacg ccgtggtgcg ttgattgact tcgagaacgg cacggtcgga 7680 cccgaagtcg aggctgccct aaagctcatg gagaaaagag agtacaaatt tgcttgccag 7740 accttcctga aagacgagat tcgtccgatg gaaaaagtac gtgctggcaa gactcgcatt 7800 gtcgacgttt tgcccgtgga acacattctt tacaccagga tgatgattgg cagattctgt 7860 gctcaaatgc acacaaacaa tggaccgcag attggctcag cggtcggttg caatcctgat 7920 gttgattggc aaagatttgg cacacatttt gctcagtaca gaaacgtgtg ggatgtggac 7980 tattcggcct ttgatgctaa ccactgcagt gacgcaatga acatcatgtt tgaggaggta 8040 tttcgcacag acttcggttt ccacccaaat gctgagtgga ttctgaagac tcttgtgaac 8100 acggagcacg cctatgagaa caaacgtatc actgttgagg gcgggatgcc gtctggctgt 8160 tccgcgacaa gcatcatcaa cacaattttg aacaacattt atgtgctcta cgctcttcgt 8220 agacactatg agggagttga gctggacacc tacaccatga tctcctacgg agatgacatc 8280 gtggttgcaa gtgactacga tctggatttt gaggctctca aaccccactt caaatctctt 8340 ggtcaaacca tcactccagc tgacaaaagc gacaaaggtt ttgttcttgg tcactccatt 8400 accgatgtca ctttcctcaa aagacacttc cacatggact atggaactgg gttttacaaa 8460 cctgtgatgg cctcaaagac cctcgaggcc attctctcct ttgcacgccg tgggaccata 8520 caggagaagt tgatctccgt ggcaggactc gccgtccact caggacctga cgagtaccgg 8580 cgtctctttg agcccttcca gggtctcttc gagattccaa gctacagatc actttacctg 8640 cgttgggtga acgccgtgtg cggtgacgca taatccctca gatgtcacaa ttggcagaaa 8700 gactctgagg cgagcgacgc cgtaggagtg aaaagcccga aagggctttt cccgcttcct 8760 attccaaaaa aaaaaaaaaa aaactagttc tagagcggcc gccaccgcgg tggagctcca 8820 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt 8880 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 8940 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 9000 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 9060 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 9120 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 9180 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 9240 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 9300 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 9360 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 9420 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 9480 gt; ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 9600 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 9660 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 9720 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 9780 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 9840 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 9900 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 9960 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 10020 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 10080 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 10140 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 10200 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 10260 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 10320 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 10380 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 10440 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 10500 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 10560 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 10620 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 10680 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 10740 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 10800 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 10860 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 10920 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 10980 aaataggggt tccgcgcaca tttccccgaa aagtgccac 11019 <210> 2 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 ggagctgggc aatccagccc ggcgac 26 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 3 ctgcttctcg ggcgcaatga tttcctg 27 <210> 4 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 4 cttctaaatt ttgacctgct caaattggcg gg 32 <210> 5 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 5 cttgagcctt ttctggacct ttgttttcca c 31

Claims (9)

Among the entire gene regions of the foot-and-mouth disease O-type vaccine strain Manisa, the gene coding for the P1 protein consisting of VP1, VP2, VP3 and VP4 as the protective antigen of the foot-and-mouth disease type O vaccine Manisa is a foot- As recombinant foot-and-mouth disease virus (hereinafter referred to as &quot; p1m-AsVNLC04-P1 &quot;) produced from the recombinant plasmid pOm-AsVNLC04-P1 having the cleavage map shown in Fig. 3, which is substituted with a gene encoding a P1 protein composed of VP1, VP2, VP3 and VP4 as,
Wherein said recombinant plasmid pOm-AsVNLC04-P1 has the nucleotide sequence of SEQ ID NO: 1,
Wherein the gene coding for the P1 protein consisting of VP1 to VP4 as the defensive antigen of the foot-and-mouth disease type 1 serotype type IV viral vaccine has a nucleotide sequence of nucleotides 2356 to 4557 of SEQ ID NO: 1. delete The recombinant foot-and-mouth disease virus according to claim 1, wherein the recombinant virus expresses the protective antigen of the foot-and-mouth disease type 1 serotype IV virus. 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 Manisa into a plasmid;
(2). In the plasmid obtained in the above step (1), the gene coding for the P1 protein consisting of VP1, VP2, VP3 and VP4 as the protective antigen of the foot-and-mouth disease type O vaccine Manisa was designated as Foot-and-mouth disease Asia with the nucleotide sequence of Gene Berkman session number GU125646 1 is replaced with a gene encoding a P1 protein composed of VP1, VP2, VP3, and VP4 as a protective antigen of a serotype IV virus, and constructing a recombinant plasmid having the recombinant gene inserted therein of SEQ ID NO: 1, A gene encoding a P1 protein consisting of VP1 to VP4 as a protective antigen of a serotype genotype IV virus vaccine has a nucleotide sequence of nucleotides 2356 to 4557 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 tongue (ZZ-R) and young hamster kidney (BHK-21) cells. [6] The method according to claim 5, wherein the recombinant virus expresses the defensive antigen of the foot-and-mouth disease type 1 serotype IV virus. 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. delete

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