KR20100121289A - Genetic recombinant classical swine fever vaccine flc-lom virus and preparing method thereof - Google Patents

Abstract

PURPOSE: A recombinant Flc-LOM swine fever vaccine virus is provided to easily detect other vaccine virus and outdoor virus and to early detect infected pig. CONSTITUTION: A vaccine virus for swine fever, Flc-LOM virus(KFCC11441P) has a nucleotide sequence of sequence number 1. In the vaccine virus, 52, 96, 220, 405th bases are changed from C, A, G, and G to T, G, T, and A. The vaccine virus Flc-LOM genetic marker or END(Exaltaton of Newcastle Disease virus). A live vaccine for swine fever contains the vaccine virus Flc-LOM virus(KFCC 11441P).

Description

Genetic recombinant classical swine fever vaccine Flc-LOM virus and preparing method

The present invention relates to a genetically engineered swine fever vaccine virus, and more particularly, to extract full-length cDNA for total RNA by extracting genomic RNA from a swine fever virus strain and using the synthesized cDNA as a T7 promoter of pACYC177 plasmid. Cloning the downstream to prepare a recombinant plasmid and the resulting recombinant plasmid is transcribed in vitro to prepare RNA, and then mixed lipofectin (lipofectin) and injected into pig kidney cells (transfection) Flc-LOM virus It relates to a method for producing a vaccine virus Flc-LOM virus, characterized in that it comprises a step of producing a vaccine virus Flc-LOM virus produced by the production method thereof.

Swine fever causes high fever, lack of appetite, diarrhea or constipation, cyanosis and hind limb misalignment or staggering. It is classified as Class A by the Secretariat (OIE) and is a malignant infectious disease that is regarded as the first kind of animal epidemic in the animal epidemic prevention method.

The swine fever pathogen is a classical swine fever virus (CSFV) belonging to the Pestivirus of Flaviviridae. The incubation period usually lasts 6-11 days, sometimes 20-30 days or longer (OIE standard: 40 days). The path of infection is mainly digestive and respiratory organs, and the invading virus proliferates in the tonsils, lymph nodes, and parenchymal cells. Sick pigs also produce large amounts of viruses. Swine fever is transmitted through various routes such as the movement of infected pigs, vehicles, livestock tools, and people (shoes and clothes). The above infectious diseases cause a loss of productivity and economic damage of the pig industry.

The live swine fever vaccine currently used in Korea uses LOM virus, which is a biological marker that contains END positive (END + ve) and END negative (END -ve) phenomena. . In addition, fixed genetic markers have not been investigated, making it difficult to distinguish them from other existing live and outdoor viruses. Therefore, in order to effectively prevent swine fever, it is efficient to use a vaccine virus fixed with genes and biological markers, and furthermore, a swine fever vaccine using domestic isolates is required.

An object of the present invention is to provide a live vaccine to prevent swine fever, and vaccine virus by distinguishing the swine fever vaccine virus and the outdoor virus that is used as a live attenuated live vaccine by genetic analysis and biomarker analysis The aim of this study is to provide a new vaccine virus that can be used to discriminate between vaccinated and outdoor infection viruses and to easily track mutations of vaccine viruses caused by genetic mutations.

The present invention to solve the above problems provides a swine fever vaccine virus Flc-LOM virus (KFCC11441P) having a nucleotide sequence of SEQ ID NO: 1 and a live swine fever vaccine comprising the same. In addition, the present invention comprises the steps of synthesizing the full-length cDNA for the entire RNA by extracting genomic RNA from the classical swine fever virus (CSFV) strain; and cloning the synthesized cDNA to the T7 promoter downstream of the pACYC177 plasmid Preparing a Flc-LOM # 5 recombinant plasmid; And preparing RNA by transducing the prepared recombinant plasmid in vitro, mixing lipofectin and transfecting pig kidney cells to produce Flc-LOM virus. It provides a method for producing a swine fever vaccine virus Flc-LOM virus (KFCC11441P).

The genetically modified swine fever live vaccine Flc-LOM virus prepared by the present invention serves as a genetic marker and a biomarker, and when used as a vaccine for preventing swine fever, facilitates differentiation from other vaccine viruses and outdoor viruses. Allow early search. Therefore, the present invention is effectively used to prevent swine fever, and to easily track the mutation of the swine fever vaccine virus to help safe management of the vaccine.

The present invention provides a swine fever vaccine virus Flc-LOM virus (KFCC11441P) having a nucleotide sequence of SEQ ID NO: 1.

In the present invention, the vaccine virus is a C, A, G, G to T, G, T, A 52, 96, 220, 405th base in the NTR (5'non translated region) gene on the basis of the entire sequence , 2608 and 5899 bases in the E2 gene (envelope protein gene) are A, A to G, G, and 5956 and 6049 bases in the NS3 gene (nonstructural protein gene 3) are G, G to A and A, respectively. Thus, the 7054th base in the NS4A gene (nonstructural protein gene 4A) is characterized in that the conversion from G to T.

In the present invention, the vaccine virus is characterized in that any one or more markers selected from Flc-LOM gene markers, END (Exaltaton of Newcastle Disease virus) biomarkers.

The present invention also provides a live swine fever vaccine comprising the swine fever vaccine virus Flc-LOM virus (KFCC11441P).

The present invention also comprises the steps of synthesizing the full-length cDNA for the entire RNA by extracting genomic RNA from the classical swine fever virus (CSFV) strain; and cloning the synthesized cDNA to the T7 promoter downstream of the pACYC177 plasmid Preparing Flc-LOM # 5 recombinant plasmid; And preparing RNA by transducing the prepared recombinant plasmid in vitro, mixing lipofectin and transfecting pig kidney cells to produce Flc-LOM virus. It provides a method for producing a swine fever vaccine virus Flc-LOM virus (KFCC11441P).

In the method of the present invention, the vaccine virus has 52, 96, 220, and 405th bases in the NTR (5'non translated region) gene on the basis of the entire nucleotide sequence, respectively, C, A, G, G, T, G, and T. , A, 2608 and 5899 bases in the E2 gene (envelope protein gene) are A, A to G, G, and 5956 and 6049 bases in the NS3 gene (nonstructural protein gene 3) are G and G, respectively. A and A, characterized in that the 7054th base in the NS4A gene (nonstructural protein gene 4A) is converted from G to T.

Hereinafter, the swine fever vaccine virus Flc-LOM virus of the present invention and a preparation method thereof will be described in more detail.

The present invention extracts the full-length genome of CSFV, a swine fever virus for the production of live attenuated swine fever vaccines containing two types of biomarkers (END + and END-), to create a full-length cDNA and generate a new gene. For preparing attenuated live vaccines in which a full-length infectious cDNA was generated, cloned, reverse transcribed into genomic RNA, and artificially transfected into porcine kidney cells to generate a genetic marker or a single biomarker (END +). Recombinant swine fever virus Flc-LOM virus and a method for producing the same.

In the present invention, in order to prepare a swine fever vaccine using the Flc-LOM virus and to distinguish it from the existing swine fever vaccine and the outdoor swine fever virus, a) swine fever virus Flc- containing a genetic marker and END biomarker Shows the manufacturing process of LOM virus, b) the Flc-LOM virus as a live swine fever vaccine to confirm immunogenicity and safety when administered to swine, and c) the swine fever virus FLc- containing genetic marker and END biomarker. When LOM virus is used as a swine fever vaccine, a process for distinguishing it from other swine fever live vaccine and field swine fever virus is presented.

The inventors of the present invention perform a virus development study for vaccines that can prevent swine fever using genetic recombination technology to produce a live vaccine for swine fever live vaccine containing a genetic marker and a bio (biological) marker and to prepare a live vaccine using the same. The present invention was completed by confirming safety and immunogenicity in pigs.

Hereinafter, embodiments of the present invention will be described. However, the following examples and the like are intended to explain the present invention in more detail and are not intended to limit the rights of the present invention.

Example 1 Preparation of Flc-LOM Virus

1. Preparation of vaccine virus Flc-LOM virus

(1) Genomic RNA was extracted from the CSFV strain, a swine fever virus, to synthesize full-length cDNA for total RNA. The synthesized cDNA was cloned into the T7 promoter downstram of pACYC177 plasmid, a vector purchased from NEB, to prepare a Flc-LOM # 5 recombinant plasmid (see FIG. 1).

The Flc-LOM # 5 was transcribed in vitro to generate Flc-LOM # 5 RNA (see FIG. 2), and after mixing the RNA with lipofectin, porcine kidney 15 (PK15). Transfection was performed. Virus was generated from the cells transfected with RNA, and it was multiplied and named Flc-LOM virus. The named virus was deposited with the Korea Microorganism Conservation Center (KCCM), and on March 20, 2009, the microorganism was named Flc-LOM virus and the microorganism accession number was KFCC11442P.

(2) Flc-LOM virus was confirmed to specifically react with the swine fever specific monoclonal antibody (3B6) (see Fig. 3), the titer was 10 ^4.25 TCID ₅₀ / ml. Subsequently, seed stock was prepared by passage up to 5 generations in pig kidney cells (PK15). The mean virus titer was found to be 10 ^5.5 TCID ₅₀ / ml, and the proliferative properties of the virus reached the highest titer at 72 hours upon inoculation with 1 moi (see FIG. 4).

(3) When the RNA gene sequence of the Flc-LOM virus is compared with that of the swine fever virus before genetic recombination, eight genes are transformed in four gene regions including NTR, E2, NS3 and NS4A. The difference from the virus was confirmed (see Table 1 and SEQ ID NO 1 below).

TABLE 1 Location of Flc-LOM virus gene changes

2. Differentiation of Flc-LOM virus with genetic markers

A primer set capable of amplifying four gene regions was prepared as shown in Table 2 below. The primer set production method is a synthesis of DNA synthesizer oligonucletide, a single stranded DNA that can be amplified by including four altered gene regions by RT-PCR (reverse transcriptase polymerase chain reaction) (see SEQ ID NO: 2-9). .

[Table 2] Primers for rt-PCR which can be confirmed by gene sequencing by amplifying Flc-LOM virus specific genes

One-step RT-PCR conditions are as follows: temperature 30min / 42 ℃, 15min / 94 ℃, (40cycles: 30sec / 94 ℃, 30sec / 53 ℃, 40sec / 72 ℃), 10min / 72 ℃ It was confirmed by electrophoresis on agarose gel (see FIG. 5). Therefore, Flc-LOM virus could be distinguished from other types of swine fever vaccine virus and field swine fever virus using RT-PCR and gene sequencing analysis.

3. Experiment of vaccinated virus Flc-LOM virus with END biomarker

RNA produced from the Flc-LOM # 5 recombinant plasmid was mixed with lipofectin and transfected into porcine kidney 15 (PK15) cells to proliferate the generated Flc-LOM virus.

The primary swine testicle cells (ST cells) were invited to 96well microplates and incubated for 24 hours. Flc-LOM virus was diluted 10-fold step by 8 replicates. Cell culture supernatants of 96 well microplates in which mononuclear piglet cells were monolayered were removed and inoculated with 100 μl of Flc-LOM virus for each dilution step. After 4 days of incubation at 37 ° C. carbonic acid incubator, all of the culture supernatant was removed and sensitized for 2 hours by adding 100 μl of Newcastle disease virus (NDV) of 10 ^4.0 PFU. In addition, 100 μl of cell culture medium was added to observe the presence of cytopathic effect after incubation for 3 days, and the biomarker of END + ve was identified. It can be seen that the virus was prepared (see FIG. 6).

<Example 2> Preparation of live swine fever vaccine containing Flc-LOM virus

1. Preparation of live swine fever vaccine containing Flc-LOM virus

Flc-LOM virus was inoculated into PK15 cells at 1 moi (multiplicity of infection) and incubated in a 5% carbon dioxide gas incubator for 72 hours, followed by freezing and thawing of the culture medium three times at room temperature and -40 ° C. All viruses propagated at were allowed to elute. Virus culture supernatant was centrifuged at 1500 rpm for 20 minutes and the supernatant was harvested and titrated on PK15 cells to use more than 1 × 10 ⁵ TCID ₅₀ / ml of the virus for vaccine production.

Vaccine production was carried out in the following manner. Mix 2x10 ^4.0 TCID ₅₀ / ml titer of Flc-LOM virus and the protective agent LPGG (lactose phophate glutamate gelatin mixture) in the same amount, so that the concentration of Flc-LOM virus is 1x10 ^4.0 TCID ₅₀ / ml and subdivided by 1ml Vaccines were prepared.

2. Safety test of manufactured live vaccine

Ten test vaccines were inoculated into four pigs 40-40 days old. As a control, sterilized phosphate buffer solution was injected into three piglets (see Table 3 below). After vaccination, the virus contents in the blood, saliva, feces and nasal juice were observed for 28 days. Vaccine virus was observed in the blood, nasal juice and feces temporarily between 5 to 10 days after inoculation. As observed, there was no difference from the normal control group, so no adverse effect was observed after Flc-LOM virus inoculation (see Table 4 below).

In addition, in the leukocyte change after Flc-LOM virus inoculation, both the inoculated group and the control group showed a normal range (10,000-30,000 / ㎣) (see FIG. 7), and the rectal temperature change was also confirmed to be normal (see FIG. 8). The safety of the Flc-LOM virus was confirmed to be excellent.

Table 3 Safety test method in pigs inoculated with Flc-LOM virus

[Table 4] Virus, Blood, Saliva, Fecal, and Nasal Virus Detection and Clinical Symptoms after Vaccination

(* In Table 4, * represents the number of positive heads / test heads.)

3. Immunogenicity Test

In order to confirm immunogenicity in pigs, 45-day-old pigs with a negative swine fever antibody were purchased, and two control mice and five mice were used as the Flc-LOM virus inoculation group (see Table 5 below). Flc-LOM virus inoculated group showed an average of 128 times of virus neutralizing antibody titer after 3 weeks of first vaccination and showed an excellent antibody titer of 256 times or more after 2 weeks of second vaccination (see FIG. 9). Two weeks after the second inoculation, the SW03 virus, a swine fever and highly pathogenic swine fever virus, was challenged by inoculation at 100 mld (minimum lethal dose) by intramuscular inoculation. % Survival was confirmed that the protective effect of the swine fever vaccine using the Flc-LOM virus is excellent (see Table 6 below).

Table 5 Experimental design of immunogenicity and protective effect in pigs inoculated with Flc-LOM virus

Table 6 Immunogenicity and Protective Effects in Pigs Inoculated with Flc-LOM Virus

The swine fever vaccine virus Flc-LOM virus produced by the present invention has a function as a genetic marker and an END biomarker, and can be used as a vaccine for preventing swine fever, thereby increasing the profitability of livestock farmers and contributing to the research and development of livestock. Ultimately, it is expected to contribute to the national industrial development.

1 shows the Flc-LOM # 5 recombinant plasmid, which is a Flc-LOM virus cDNA clone.

Figure 2 shows the results through the electrophoresis of full-length genomic RNA generated by transcription of the Flc-LOM virus cDNA clone (Flc-LOM # 5 recombinant plasmid) in vitro .

Figure 3 shows the Flc-LOM generated after transfection of full-length genomic RNA generated by transcription of Flc-LOM # 5 recombinant plasmid in vitro with lipofectin into PK-15 cells. The virus was identified as a swine fever specific monoclonal antibody.

4 shows that the proliferative properties of Flc-LOM virus were confirmed.

Figure 5 shows that the gene was amplified using primers for identifying the Flc-LOM gene marker (G1.G2.G3, G4).

Fig. 6 shows the effect of Flc-LOM virus on END + cytopathic effect. (Picture on the left shows normal pig testicular cells and on the right shows pig testicular cells infected with Flc-LOM virus (END +).)

Figure 7 shows the changes in leukocyte count after Flc-LOM virus inoculation.

Figure 8 shows rectal body temperature change after Flc-LOM virus inoculation.

Figure 9 shows the change in antibody formation after inoculation of Flc-LOM virus and the change of antibody after challenge (38 days old).

<110> National veterinary research and quarantine service <120> Genetic recombinant classical swine fever vaccine Flc-LOM virus          and preparing method <160> 9 <170> KopatentIn 1.71 <210> 1 <211> 12298 <212> DNA <213> Flc-LOM virus <400> 1 gtatacgagg ttagttcatt ctcgtatgca tgattggaca aattaaaatt ttaatttgga 60 tcagggcctc cctccagcga cggccgaact gggctggcca tgcccgcagt aggactagca 120 aacggaggga ctagccgtag tggcgagctc cctgggtggt ctaagtcctg agtacaggac 180 agtcgtcagt agttcgacgt gagcagaagc ccacctcgat atgctatgtg gacgagggca 240 tgcccaagac acaccttaac cctagcgggg gtcgctaggg tgaaatcaca ccacgtgatg 300 ggagtacgac ctgatagggt gctgcagagg cccactatta ggctagtata aaaatctctg 360 ctgtacatgg cacatggagt tgaatcattt tgaactttta tacgaaacaa acaaacaaaa 420 accaaaggga gtggaggaac cggtatacga tgccacgggg aaaccattgt ttggagaccc 480 gagtgaggta cacccacaat caacactgaa gctaccacat gataggggga gaggtaacat 540 caaaacaaca ctgaagaacc tacctaggaa aggcgactgc aggagtggca accatctagg 600 cccggttagc gggatatatg taaagcccgg ccctgtcttt tatcaggact 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taaaccaacc aagcttatga gtggaataca aacagtctcc aaaagtacca cagacttgac 5760 agaaatggta aagaaaataa cgaccatgaa caggggagaa ttcagacaaa taacccttgc 5820 tacaggtgcc ggaaaaacca cggaacttcc taggtcggtc atagaagaga tagggaggca 5880 taagagagtc ttggtcttga tccctcttag ggcggcagca gagtcagtat accagtatat 5940 gagacaaaaa catccaagca tcgcatttaa cctgaggata ggggagatga aggaagggga 6000 catggccaca gggataacct atgcctcata cggttacttc tgtcagatac cacaacctaa 6060 gttgcgagcc gcaatggttg agtactcctt catatttctt gatgagtacc actgcgccac 6120 cccagaacaa ttggctatca tgggaaagat ccacagattt tcagagaacc tgcgggtagt 6180 agccatgacc gcaacaccag caggcacggt aacaaccaca gggcagaaac accctataga 6240 agaattcata gccccagaag tgatgaaagg ggaagactta ggctcagagt acttggacat 6300 tgctggacta aagatacctg tagaggagat gaagagcaac atgctggttt ttgtgcccac 6360 taggaacatg gcggtggaga cagcaaagaa attgaaagct aagggctaca actcaggcta 6420 ctattatagt ggagaggatc catctaacct gagagtggta acgtcacagt ccccatacgt 6480 ggtggtggca accaacgcga tagaatcagg tgttactctc ccggacttag atgtggtcgt 6540 cgatacaggg cttaagtgtg aaaagagaat acggctgtca actaagatgc ccttcatagt 6600 gacgggcctg aagaggatgg ctgtcacgat tggggaacaa gcccagagaa gggggagagt 6660 tgggagagta aagcctggga gatactacag gagtcaagaa actcccgttg gttctaaaga 6720 ttaccattat gatctactgc aagcacagag gtacggtatt gaagatggga taaacatcac 6780 caaatccttt agagagatga actatgattg gagcctttat gaggaggaca gtctgatgat 6840 tacacaattg gaaatcctca ataatttgtt gatatcagaa gaactaccga tggcagtaaa 6900 aaatataatg gccaggactg accacccaga accaattcag ctggcgtaca acagctacga 6960 aacacaagtg ccagtgctat tcccaaaaat aaaaaatgga gaggtgactg acagttacga 7020 taactatacc ttcctcaacg caagaaaatt gggtgatgat gtaccccctt acgtgtatgc 7080 cacagaggat gaggacttag cggtagagct gctgggctta gactggccag accctggaaa 7140 ccaaggaacc gtagaggctg gcagagcact aaaacaagta gttggtctat caacagctga 7200 gaatgccctg ttagtagcct tattcggcta tgtaggatat caggcacttt caaagaggca 7260 tataccagta gtcacagata tatattcaat tgaagatcac aggttggaag acaccacaca 7320 cctacagtac gccccgaatg ctatcaagac ggaggggaag gagacagagt tgaaagagct 7380 agctcagggg gatgtgcaga gatgtgtgga agctatgacc aattatgcaa gagagggcat 7440 ccagttcatg aagtctcagg cactggaggt gaaagaaacc cccacttaca aagagacaat 7500 ggacactgtg acggactatg taaagaaatt catggaggcg ctgacagaca gtaaagaaga 7560 catcataaga tatgggttgt gggggacgca cacggcccta tataagagca tctgtgccag 7620 gctcgggagt gagactgcgt tcgctaccct ggttgtgaag tggctggcat ttggggggga 7680 atcaatagca gaccatgtca aacaagcggc cacagacttg gtcgtttact atatcatcaa 7740 cagacctcag ttcccaggag acacggagac acaacaggaa ggaaggaaat ttgtggccag 7800 cctaatggtc tcagctctag ttacttacac atacaaaagc tggaattaca ataatctgtc 7860 caagatagtt gaaccggcct tagccactct gccctatgcc gccacagctc tcaaactatt 7920 cgcccccact cgattggaga gcgttgtcat attgagtacc gcaatctaca aaacctacct 7980 gtcaatcagg cgcggaaaaa gcgatggttt gctaggcaca gggattagtg cggctatgga 8040 gatcatgtca caaaatccag tatccgtggg catagcagtc atgctagggg taggggccgt 8100 ggcagcccac aatgcaatcg aagccagtga gcagaagaga acactactca tgaaagtttt 8160 tgtaaagaac ttcttggacc aggcagccac tgatgaatta gtcaaggaga gtcctgagaa 8220 aataataatg gctttgtttg aagcagtgca gacagtcggc aaccctctta gactagtata 8280 ccacctttat ggagtttttt ataaggggtg ggaggcaaaa gagttggccc aaaggacagc 8340 cggtaggaac cttttcactt tgataatgtt cgaggctgtg gaactactag gagtagatag 8400 tgaaggaaag atccgccagc tatcaagtaa ttacattcta gagctcctgt ataagttccg 8460 tgacagtatc aagtctagcg tgagggagat ggcaatcagc tgggcccctg cccctttcag 8520 ttgtgattgg acaccgacgg atgacagaat agggctcccc caagacaatt tcctccaagt 8580 ggagacgaaa tgcccctgtg gttacaagat gaaggcagtt aagaattgtg ctggagagct 8640 aagactctta gaggaggaag gctcatttct ctgcagaaat aaattcggga gaggttcacg 8700 gaactacagg gtgacaaaat actatgatga caatctatca gaaataaagc cagtgataag 8760 aatggaaggg catgtggaac tatactacaa gggggccaca atcaaactgg atttcaacaa 8820 cagtaaaaca atattggcaa ccgataaatg ggaggttgat cactccactc tggtcagggt 8880 gctcaagagg cacacagggg ctggatatca tggggcatac ctgggcgaga aaccgaacca 8940 caaacatctg atagagaggg actgtgcaac catcaccaaa gataaggttt gttttctcaa 9000 aatgaagaga gggtgtgctt tcacttatga cttatccctt cacaacctta cccgactgat 9060 tgaattggta cacaagaata acttggaaga caaagagatc cctgctgtta cggttacaac 9120 ctggctggct tacacgtttg taaatgaaga tatagggacc ataaaaccag ccttcgggga 9180 gaaagtaaca ccggagatgc aggaggagat aaccttgcag cctgctgtag tggtggatac 9240 aactgacgtg accgtgactg tggtagggga agcccctact atgactacag gggagactcc 9300 gacagcgttc accagctcag gttcagaccc gaaaggccaa caagttttaa aactgggggt 9360 aggtgaagga caataccccg ggatcaatcc acagagggca agcctgcacg aagccataca 9420 aggtgctgat gagaggccct cggtgctgat attggggtct gataaagcca cctctaatag 9480 agtaaaaact gcaaagaatg taaaggtata cagaggcagg gacccactag aagtgagaga 9540 tatgatgagg aggggaaaga tcctggtcgt agccctgtct agggttgata atgccctatt 9600 gaaatttgtt gattacaaag gcacctttct aactagagag accctagagg cattaagttt 9660 gggtaggcct aaaaagaaaa acataaccaa ggcagaagca cagtggttgc tgtgtcttga 9720 agaccaaatg gaagagctac ccgattggtt cgcggccggg gaacccattt ttctagaggc 9780 caacattaaa catgacaggt atcatctggt gggggatata gctactatca aggaaaaagc 9840 caaacagttg ggggctacag actccacaaa gatatctaag gaggttggtg caaaagtgta 9900 ttctatgaaa ctgagtaatt gggtgatgca agaagaaaat aaacagggca acctgacccc 9960 cttgttcgaa gagctcctgc aacagtgtcc acccgggggc cagaacaaaa ctgcacatat 10020 ggtctctgct taccaactag cccaagggaa ctggatacca accagctgcc atgtttttat 10080 ggggaccata tctgccagga ggaccaagac ccatccatat gaagcatatg tcaagttaag 10140 ggagttggta gaggaacaca agatgaaaac attgtgtccc ggatcaagcc tgggtaagca 10200 caacgaatgg ataattggta agatcaaata ccagggaaac ctgaggacca aacacatgtt 10260 gaaccccggc aaggtggcag agcaactgtg cagagaggga cacagacaca atgtgtataa 10320 caagacaata ggttcagtaa tgacagctac tggtatcagg ttggagaaat tgcccgtggt 10380 tagggcccag acagacacaa ccaacttcca ccaagcaatc agggataaga tagacaagga 10440 agagaaccta cagaccccgg gtttacataa gaaactaatg gaggttttca atgcattgaa 10500 acgacccgag ttagagtcca cctacgatgc cgtggaatgg gaggaactgg agagaggaat 10560 aaacaggaag ggtgctgctg gtttcttcga acgcaaaaat ataggggaaa tattggattc 10620 agagaaaaac aaagtcgaag agattattga caatctgaaa aaaggtagaa atatcaaata 10680 ctatgaaact gcgatcccaa agaatgagaa gagggacgtc aatgatgact ggacctctgg 10740 tgacttcgtg gacgagaaga agcccagagt catacaatac cctgaagcaa aaacaaggct 10800 ggccatcacc aaggtgatgt ataagtgggt gaagcagaag ccagtagtta tacccgggta 10860 tgaagggaag acacctctgt tccaaatttt tgacaaagta aagaaggaat gggatcaatt 10920 ccaaaatcca gtggcagtga gcttcgacac taaggcgtgg gacacccagg taaccacaaa 10980 agatttggag ctgataaagg acatacaaaa gtactatttc aagaagaaat ggcataaatt 11040 tattgacacc ctgaccatgc atatgtcaga agtacccgta atcagtgccg atggggaagt 11100 atacataagg aaagggcaaa gaggcagtgg acaacctgac acaagcgcag gcaatagcat 11160 gctaaatgtg ttaacaatgg tttacgcctt ctgcgaggcc acgggagtac cctacaagag 11220 ctttgacagg gtggcaaaaa ttcatgtgtg cggggatgat ggtttcctga tcacagaaag 11280 ggctctcggt gagaaattcg cgagcaaggg agtccagatc ctatatgaag ctgggaagcc 11340 ccagaagatc actgaagggg ataaaatgaa attggcctac caatttgatg atattgagtt 11400 ttgctcccat acaccaatac aagtaaggtg gtcagataac acctctagtt acatgccggg 11460 gagaaataca accacaatcc tggctaaaat ggccacaagg ttagattcca gtggtgagag 11520 gggcaccata gcatatgaga aagcagtagc attcagcttc ctcctgatgt actcctggaa 11580 cccactaatt agaaggatct gcttactggt gctatcaact gaactgcaag tgaaaccagg 11640 gaagtcaact acttactact atgaagggga cccgatatct gcctacaagg aagtcatcgg 11700 ccacaatctt tttgatctca agagaacaag cttcgagaag ctggccaaat taaatctcag 11760 catgtctgta ctcggggctt ggacaagaca caccagcaaa agactattac aagactgtgt 11820 caatatgggt gttaaagagg gcaactggct agttaatgca gacagactag tgagtagcaa 11880 gactggaaac aggtacatac ctggagaggg ccacaccctg caagggagac attatgaaga 11940 actggtgttg gcaagaaaac agattaataa ctttcaaggg acagacaggt acaatctagg 12000 cccaatagtc aacatggtgt taaggaggct gagagtcatg atgatgaccc tgatagggag 12060 aggggtatga acgcgggcaa cccgggatct ggacccgcca gtaggaccct attgtaaata 12120 acactaattt tttatttatt tatatattat tatctattta tttatttatt tattgaatga 12180 gtaagaactg gtacaaacta cctcaagtta ccacactaca ctcattttta acagcacttt 12240 agctggaagg aaaattcctg acgtccacag ttggactaag gtaatttcct aacggccc 12298 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rt-PCR of Flc-LOM virus <400> 2 gtatacgagg ttagctcatt 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rt-PCR of Flc-LOM virus <400> 3 gtttccccgt ggcatcgtat 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rt-PCR of Flc-LOM virus <400> 4 agaccagact ggtggccata tga 23 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rt-PCR of Flc-LOM virus <400> 5 tttaccactt cagttctca 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rt-PCR of Flc-LOM virus <400> 6 tcctaggtcg gtcatagaag 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rt-PCR of Flc-LOM virus <400> 7 ccaagtactc tgagcctaag 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rt-PCR of Flc-LOM virus <400> 8 taatggccag gactgaccac 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rt-PCR of Flc-LOM virus <400> 9 cctccgtctt gatagcattc 20  

Claims (6)

Porcine fever vaccine virus Flc-LOM virus (KFCC11441P) having the nucleotide sequence of SEQ ID NO: 1. According to claim 1, wherein the vaccine virus is 52, 96, 220, 405th base in the NTR gene on the basis of the entire sequence C, A, G, G to T, G, T, A, respectively, in the E2 gene 2608 and 5899 bases in A, A to G, G, 5956 and 6049 bases in NS3 gene, respectively, G, G to A, A, and 7054 bases in NS4A gene, G to T Porcine fever vaccine virus Flc-LOM virus. The swine fever vaccine virus Flc-LOM virus according to claim 1, wherein the vaccine virus is any one or more markers selected from among Flc-LOM gene markers and END biomarkers. The live swine fever vaccine comprising the swine fever vaccine virus Flc-LOM virus (KFCC11441P) of any one of claims 1 to 3. Extracting genomic RNA from a classical swine fever virus (CSFV) strain to synthesize full-length cDNA for total RNA; Cloning the synthesized cDNA to the T7 promoter downstream of the pACYC177 plasmid to produce a Flc-LOM # 5 recombinant plasmid; And Preparing the RNA by transcribing the prepared recombinant plasmid in vitro, mixing lipofectin and transfecting pig kidney cells to produce Flc-LOM virus; Method for producing swine fever vaccine virus Flc-LOM virus (KFCC11441P). 6. The vaccine virus of claim 5, wherein the 52, 96, 220, and 405th bases in the NTR gene are each C, A, G, G to T, G, T, A in the E2 gene. 2608 and 5899 bases in A, A to G, G, 5956 and 6049 bases in NS3 gene, respectively, G, G to A, A, and 7054 bases in NS4A gene, G to T Method for producing a swine fever vaccine virus Flc-LOM virus, characterized in that.

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