KR20100121288A - Genetic recombinant classical swine fever vaccine flc-lom-berns virus and preparing method thereof - Google Patents

Abstract

PURPOSE: A vaccine virus for swine fever of gene recombination is provided to easily detect mutation of vaccine virus and to early remove infected pig. CONSTITUTION: A vaccine virus for swine fever, Flc-LOM-BErns virus(KFCC11442P) contains a nucleotide of sequence number 1. An Erns gene of CSFV(classical swine fever virus) is substituted with Erns gene of BVDV(bovine viral disease virus) in the vaccine virus. The vaccine virus is Erns gene marker, Erns serologic marker or END(Exaltaton of Newcastle Disease virus) bio marker. A method for manufacturing swine vaccine virus, Flc-LOM-BErns virus(KFCC11442P) comprises: a step of extracting genomic RNA from swine fever virus strain; a step of synthesizing full-length cDNA to total RNA; a step of removing Erns gene from the synthesized cDNA; and a step of inserting Erns gene of BVDV.

Description

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

The present invention relates to a genetically engineered swine fever vaccine virus, and more particularly, to extract genomic RNA from a swine fever virus strain to synthesize full-length cDNA for the entire RNA and to remove the Erns gene from the synthesized cDNA. The present invention relates to a swine fever vaccine virus Flc-LOM-BErns virus produced by the step of preparing a cDNA containing BVDV Erns gene by inserting the Erns gene of BVDV.

Swine fever causes swine fever, appetite deficiency, diarrhea or constipation, cyanosis and hind limb misalignment or staggering.The swine fever is the most feared disease of swine disease where there is no cure and all infected pigs die. 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 classic swine fever virus (CSFV) belonging to the Pestivirus of Flaviviridae. The path of infection is mainly digestive and respiratory. Placenta infection is also possible. 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. Swine fever live vaccine currently used in Korea is not distinguished serologically from LOM strain (strain), or vaccines using other existing strains and field pathogenic swine fever virus. Therefore, in order to effectively prevent swine fever, there is a need for a method for efficiently testing outdoor infections and vaccinations by testing the blood of pigs that are shipped to slaughterhouses without directly visiting infected farms. The most appropriate method for this purpose is to replace some genes of swine fever virus with a modified formula to prevent the production of antibodies originally generated from swine fever virus infection. It is to develop a credit virus.

An object of the present invention to prevent swine fever and to provide a live vaccine for the production of live vaccines that can be distinguished serologically through an antibody test with outdoor pathogenic viruses and existing live vaccines.

Another object of the present invention is to clone and analyze the whole gene of the classical swine fever virus (CSFV) LOM strain, which is being used as a live attenuated live vaccine, and to generate a gene marker (CSFV Erns deficiency and BVDV Erns positive). ) And a single recombinant swine fever vaccine virus (Flc-LOM-BErns virus) with biomarkers (END) generated. The purpose of the present invention is to provide a method for preparing a vaccine that can distinguish an outdoor infection.

In order to solve the above problems, the present invention provides a swine fever vaccine virus Flc-LOM-BErns virus (KFCC11442P) 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 total RNA by extracting genomic RNA from the swine fever virus (CSFV) strain; And removing the Erns gene from the synthesized cDNA, and inserting the Erns gene of BVDV KD26-1 to prepare a cDNA including the BVDV Erns gene. Flc-LOM-BErns It provides a method for producing a virus (KFCC11442P).

The recombinant Flc-LOM-BErns virus prepared by the present invention functions as an Erns gene marker, an Erns serologic marker, and an END biomarker. Since the mutation of the fever vaccine virus can be easily tracked, it can be effectively used to prevent and treat swine fever by detecting and removing pigs infected with the swine fever field virus early through serological and genetic discrimination.

The present invention provides a swine fever vaccine virus Flc-LOM-BErns virus (KFCC11442P) having a nucleotide sequence of SEQ ID NO: 1.

In the present invention, the vaccine virus is characterized in that the Erns gene of CSFV (classical swine fever virus) is replaced with the Erns gene of Bovine Viral Disease Virus (BVDV).

In the present invention, the vaccine virus is characterized in that any one or more markers selected from Erns genetic markers, Erns serological markers and END (Exaltaton of Newcastle Disease virus) biomarkers.

The present invention also provides a classic swine fever live vaccine comprising the swine fever vaccine virus Flc-LOM-BErns virus (KFCC11442P).

The present invention also comprises the steps of synthesizing the full-length cDNA for the total RNA by extracting genomic RNA from the swine fever virus (CSFV) strain; And removing the Erns gene from the synthesized cDNA, and inserting the Erns gene of BVDV to prepare a cDNA including the BVDV Erns gene. Flc-LOM-BErns virus (KFCC11442P) It provides a method of manufacturing).

The production method of the present invention clones the prepared cDNA into a plasmid to produce a Flc-LOM-BErns virus recombinant plasmid, and transduced in vitro to prepare RNA and then mixed lipofectin (lipofectin) porcine kidney cells It is characterized in that the further step of preparing the Flc-LOM-BErns virus by transfection.

Hereinafter, the swine fever vaccine virus Flc-LOM-BErns 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 virus, and makes it into a full-length cDNA, and then removes the Erns gene of the cDNA and the small viral diarrheal virus Erns gene. After replacement, the recombinant full-length infectious cDNA was prepared and cloned, and then transcribed into genomic RNA in vitro and transfected into porcine kidney cells to remove the CSFV Erns gene and insert the Erns gene of BVDV. The present invention relates to a Flc-LOM-BErns virus, a genetically modified swine fever vaccine virus for producing an attenuated live vaccine, in which a genetic marker, an Erns serological marker, or an END biomarker is generated in cell culture.

In the present invention, in order to prepare a swine fever vaccine using the prepared Flc-LOM-BErns virus and to distinguish it from the existing swine fever vaccine and the outdoor swine fever virus, a) pigs containing a genetic marker and END biomarker Flc-LOM-BErns virus shows the manufacturing process, b) Flc-LOM-BErns virus produced a live swine fever vaccine to confirm the immunogenicity and safety when administered to pigs, c) CSFV Erns protein is missing And swine fever vaccine virus Flc-LOM-BErns virus with END biomarker, when used as a swine fever vaccine to distinguish from other swine fever live vaccines by genetic testing and d) Erns serum lacking CSFV Erns protein Other swine fever live vaccine virus when used as swine fever vaccine using the swine fever vaccine virus Flc-LOM-BErns virus, Et al shows a process to distinguish serologically through the antibody test for swine fever virus and CSFV Erns protein.

The present inventors carried out a research on the development of a vaccine virus for the prevention of swine fever using genetic recombination technology. Therefore, the Erns gene of the antiviral diarrhea virus which isolated the Erns gene of the swine fever vaccine virus isolated in Korea. The present invention was completed by preparing a live vaccine virus for swine fever live vaccine by using the same, and forming an antibody for swine fever Erns protein by replacing with an antibody, thereby confirming safety and immunogenicity in swine.

Hereinafter, embodiments of the present invention will be described. However, the following examples and the like are intended to specifically describe the present invention and are not intended to limit the rights of the present invention.

Example 1 Preparation of Flc-LOM-BErns Virus

1. Preparation of vaccine virus generated by replacing the Erns gene of CSFV with Erns gene of BVDV

(1) Genomic RNA was extracted from a swine fever virus (CSFV) strain to synthesize full-length cDNA for total RNA. BVDV Erns gene was included by removing Erns gene (735bp) from synthesized cDNA and inserting Erns gene (735bp) of bovine viral diarrhea virus (BVDV) KD26-1 strain isolated in Korea CDNA was prepared (see FIG. 1).

The Erns gene is a common gene of the pestivirus genus, in which swine fever virus and bovine viral diarrheal virus belong to the virus taxonomy, constituting an envelope of the virus, and having RNase activity and pathogenicity. In addition, the BVDV KD26-1 is a virus isolated in Korea and used as a bacterium in the National Veterinary Research and Quarantine Service, and the gene sequence is as described in SEQ ID NO: 2.

The cDNA was cloned into the T7 promoter downstram of pACYC177 plasmid purchased from NEB to prepare a Flc-LOM-BErns virus recombinant plasmid (see FIG. 2).

RNA was generated by transcription of the Flc-LOM-BErns virus recombinant plasmid in vitro (see FIG. 3), and the RNA was mixed with lipofectin and injected into porcine kidney cells (porcine kidney 15: PK15) cells. (transfection). Virus was generated from cells transfected with RNA, and it was propagated and named as Flc-LOM-BErns virus, and it was deposited with the Korea Microorganism Conservation Center (repaired on March 20, 2009. The name of the microorganism is Flc- LOM-BErns virus, microbial accession number is KFCC11442P).

(2) Flc-LOM-BErns virus was confirmed to specifically react with the swine fever specific monoclonal antibody (3B6) (see Fig. 4), the titer was 10 ^4.75 TCID ₅₀ / ml. Subsequently, virus seed stocks were 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 their highest titers at 72 hours upon inoculation with 1 moi (see FIG. 5).

(3) Erns gene (735bps) of BVDV KD26-1 isolated in Korea instead of Erns gene (735bp) of the conventional swine fever virus (CSFV) by analyzing the RNA gene sequence of the Flc-LOM-BErns virus (Fig. 6 and SEQ ID NO: 2). In addition, a substitution position is as Table 1 below.

[Table 1] Erns gene substitution position of Flc-LOM-BErns virus

Substitution target
Gene name Gene used for substitution Substitution gene position
(Based on Flc-LOM BErns RNA sequence) Remarks Erns BVDV KD26-1
Erns (735 bp) 1115 ～ 1849

2. Differentiation of Flc-LOM-BErns Virus Using Substituted BVDV Erns Gene

A primer set (see SEQ ID NOs: 3 and 4) for identifying BVDV KD26-1 Erns gene inserted into Flc-LOM-BErns virus by PCR method was prepared as shown in Table 2 below.

[Table 2] Primer for RT-PCR which can be confirmed by gene sequencing by specifically amplifying Erns gene of Flc-LOM-BErns virus

primer
designation Primer Sequence Amplification position
(Flc-LOM-BErns
Base sequence) Amplification
(bp) BErns F-5 'ggaagaaatta-gaaaaagcattgctggcatgggcaat 3'
R-5 'gatagggcata-tgctccaaaccacgtcttactc 3' 1115-1140
1828-1849 735

One-step RT-PCR conditions are as follows: temperature 30min / 42 ℃, 15min / 94 ℃, (40cycles: 60sec / 94 ℃, 60sec / 53 ℃, 60sec / 72 ℃), 10min / 72 ℃ It was confirmed by electrophoresis on agarose gel (see FIG. 7). Therefore, RT-PCR and gene sequencing were used to distinguish between swine fever vaccine virus Flc-LOM-BErns virus and other swine fever vaccine viruses and field swine fever virus.

3. Identification of Flc-LOM-BErns virus expressing BVDV Erns protein

Cell culture supernatants were removed after incubation for 24 hours so that monolayer cultured porcine kidney 15 (PK15) monolayer cultured in 96well microplate and inoculated with 100 μl of diluted Flc-LOM-BErns virus. After incubation for 4 days in a 37 ℃ carbonic acid cut incubator, all the culture supernatant was removed, and 100 μl of Aceton / methanol (1: 1) solution was added to 96well and fixed for 5 minutes. After removal of the fixative, anti-CSFV E2 mab that specifically reacts with swine fever virus and anti-BVDV Erns mab that specifically reacts with BVDV Erns protein were reacted at room temperature for 1 hour. After washing twice with 10 mM phosphate buffer (pH 7.2), the biotin-labeled anti-mouse IgG was reacted at room temperature for 30 minutes, and then the Avidin-biotin complex HRP conjugate was further reacted at room temperature for 30 minutes. Pigment kidney cells proliferated with Flc-LOM-BErns virus were developed using DAB (diaminobenzidin) to stain anti-CSFV E2 mab and anti-BVDV Erns mab. Specific staining on mab and anti-BVDV Erns mab confirmed that the BVDV Erns gene is correctly substituted and expressed as Erns protein (see FIG. 8).

4. Identification of the vaccine virus Flc-LOM-BErns virus in which the END biomarker was produced

The experiment was performed to determine the presence or absence (+ ve, -ve) of the cytopathic effect (CPE) of swine blast cells caused by the END (Exaltaton of Newcastle Disease virus) phenomenon of Flc-LOM-BErns virus.

The primary swine testicle cells (ST cells) were invited to 96well microplates and incubated for 24 hours. Flc-LOM-BErns virus was diluted 10-fold step by 8 repetitions. Cell culture supernatants of 96 well microplates in which mononuclear piglet cells were monolayered were removed and inoculated with 100 μl of Flc-LOM-BErns virus for each dilution step. 37 ℃ by the Newcastle disease virus (NDV) of 10 ^4.0 PFU after removing all of the culture supernatant after four days incubation in a carbon dioxide incubator cutlet added in 100㎕ were sensitized for 2 hours. In addition, 100 μl of cell culture medium was added to observe the presence of cytopathic effect after incubation for 3 days. As a result, a biomarker of END + ve was confirmed. In addition, the Flc-LOM-BErns virus of the present invention showed a biomarker of END-ve and showed different characteristics from the conventional one (see FIG. 9).

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

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

Flc-LOM-BErns 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 three times at room temperature and -40 ° C. All viruses propagated in PK15 cells were eluted. Virus culture supernatant was centrifuged at 1500 rpm for 20 minutes and the supernatant was harvested and titrated on PK15 cells to use 1 × 10 ^5.5 TCID ₅₀ / ml or more 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-BErns virus with the same amount of protective agent LPGG (lactose phophate glutamate gelatin mixture) to mix the concentration of Flc-LOM-BErns virus to 1x10 ^4.0 TCID ₅₀ / ml And lyophilized to prepare a vaccine.

2. Safety test of the manufactured vaccine

Ten test vaccines were inoculated with 5 pigs 40-50 days old. As a control, sterilized phosphate buffer solution was injected into 4 piglets (see Table 3 below). After vaccination, the virus contents in blood, feces and nasal juice were observed for 21 days. Vaccine virus was temporarily observed in blood, nasal juice and feces between 5-10 days after vaccination, but side effects such as clinical manifestations were observed. (See Table 4 below).

In addition, in the observation of leukocyte change after inoculation with Flc-LOM-BErns virus, both the inoculation group and the control group showed a normal range (10,000-30,000 / 0,000) (see FIG. 10), and the rectal temperature change was also confirmed to be normal (FIG. 11). Flc-LOM-BErns virus was confirmed to have excellent safety.

Table 3 Safety test method of vaccine virus Flc-LOM-BErns virus

Table 4 Results of virus and saliva, fecal and nasal virus detection and clinical symptoms after vaccination with Flc-LOM-BErns virus

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

3. Immunogenicity and Protective Effect Measurement Test

In order to confirm immunogenicity in pigs, five 45-day-old pigs with a negative swine fever antibody were used in two vaccinated groups and two control groups (see Table 5 below). Flc-LOM-BErns virus inoculated group showed an average of 128 times of virus neutralizing antibody titer after 3 weeks of first vaccination and an average of 256 times or more after 2 weeks of second vaccination (see Table 6 below). Two weeks after the second inoculation, SW03 strain, a swine fever strong hospital virus, was challenged with 100MLD muscle, and all of the control groups died. However, the Flc-LOM-BErns virus inoculated group survived 100%, using Flc-LOM-BErns virus. It was confirmed that the protective effect of the swine fever vaccine is excellent (see Fig. 12).

TABLE 5 Immunogenicity and Protective Effects of Pigs Flc-LOM-BErns Virus Inoculated

Table 6 Experimental results confirming immunogenicity and protective effect in pigs inoculated with Flc-LOM-BErns virus

4. Vaccine virus (Flc-LOM-BErns virus) vaccination and field infection screening by Erns antibody screening

Pigs inoculated with the vaccine virus Flc-LOM-BErns virus do not form antibodies to the swine fever virus Erns protein, but only form antibodies to the BVDV Erns protein. Viral infection and Flc-LOM-BErns virus vaccinated individuals were distinguished by serological antibody test.

Serum obtained after inoculation with the Flc-LOM-BErns virus vaccine and serum obtained after inoculation with the outdoor swine fever virulence virus were tested for swine fever Erns antibodies by the Erns antibody differential ELISA test. Swine fever vaccine virus using Flc-LOM-BErns virus was able to distinguish between vaccination and field infection by serological examination (see Table 7 below).

[Table 7] Serological Differentiation after Inoculation of Flc-LOM-BErns Virus in the Swine Fever Vaccine

The genetically engineered swine fever vaccine virus Flc-LOM-Erns virus produced by the present invention contributes to the development and development of livestock farms by providing vaccine treatment for new swine fever, which is more efficient and selectable than existing vaccines. Ultimately, it is expected to contribute to the national industrial development.

Figure 1 shows a schematic diagram of the gene composition of the Flc-LOM BErns virus inserted the BVDV Erns gene.

2 shows a Flc-LOM-BErns virus cDNA clone.

Figure 3 shows the full-length genomic RNA generated by transcriptional transcription of the full-length genomic Flc-LOM-BErns virus cDNA clone in vitro (electrophoresis).

Figure 4 is a swine fever specific monoclonal antibody of the Flc-LOM-BErns virus generated after transfection of full-length genomic RNA generated by transcription in vitro to lipofectin in PK-15 cells (Pictures on the left show normal pig kidney cells and pictures on the right show pig kidney cells infected with Flc-LOM-BErns virus.)

5 shows that the growth characteristics of the Flc-LOM-BErns virus was confirmed.

Figure 6 shows the nucleotide sequence (SEQ ID NO: 2) of the BVDV KD26-1 Erns gene.

Figure 7 shows the picture confirmed by electrophoresis of the BVDV Erns gene generated using the Flc-LOM-BErns virus gene primer.

Figure 8 shows the figure confirming the presence or absence of Erns protein expression of Flc-LOM-BErns virus (using anti-BVDV Erns mab that specifically reacts with anti-CSFV E2 mab and BVDV Erns protein).

Figure 9 shows a picture confirming the negative (END -ve) of the END cytopathic effect of Flc-LOM-BErns virus.

Figure 10 shows the change in leukocyte count after Flc-LOM-BErns virus inoculation.

11 shows the rectal body temperature change of pigs after Flc-LOM-BErns virus inoculation.

12 shows changes in antibody formation after inoculation of Flc-LOM-BErns virus and changes in antibody after challenge (38 days old).

<110> National veterinary resaerch and quarantine service <120> Genetic recombinant classical swine fever vaccine Flc-LOM-BErns          virus and preparing method <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 12298 <212> DNA <213> Flc-LOM-BErns virus <400> 1 gtatacgagg ttagctcatt 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 acatgggccc 660 ggcctaccat agagcccctc tagagttttt tagcgaagcg cagttttgtg aggtgaccaa 720 aaggataggt agggtgacag gtagtgacgg aaggctttac catatatatg tgtgcatcga 780 tggttgcata ctgctgaagc tagccaagag gggcgagcca agaaccctga agtggattag 840 aaattccacc gactgcccat tgtgggttac cagttgctct gatgatggcg caagtggaag 900 taaagagaag aagccagata ggatcaacaa gggtaaatta aaaatagccc caaaagagca 960 tgagaaggac agcagaacta agccgcctga cgctacgatc gtagtggaag gagtaaaata 1020 ccaggtcaaa aagaaaggta aagttaaagg aaagaatacc caagacggcc tgtaccacaa 1080 taagaataaa ccaccagaat ctaggaagaa attagaaaaa gcattgctgg catgggcaat 1140 aataactata gttttgtttc aagttacaat gggagaaaac ataacacagt ggaacctaca 1200 agacaatggg acggaaggga tacaacgggc aatgtttcaa aggggtgtga atagaagtct 1260 acatggaatc tggccagaga aaatctgtac tggtgtccct tcccatctag ccaccgatat 1320 ggaactaaaa acaatccatg gtatgatgga tgcaagtgag aagaccaact acacgtgttg 1380 cagacttcaa cgccatgagt ggaacaagca tggttggtgc aactggtaca atattgaacc 1440 ctggattcta gtcatgaata gaacccaagc caatcttact gagggacaac 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atggtgttgt 3180 aatcagcaca gaggggagtc atgagtgctt gatcggtaac acaaccgtca aggtgcatgc 3240 atcagatgaa agactgggcc ccatgccatg cagacctaaa gagaccgtct ctagtgaagg 3300 acctgtaagg aaaacttcct gtacattcaa ctacgcaaaa actttgaaga acaagtacta 3360 tgagcccagg gacagctatt tccagcaata tatgcttaag ggcgagtatc agtactggtt 3420 tgacctggac gtgactgacc gccactcaga ttacttcgca gaatttgttg tcttggtggt 3480 ggtagcactg ttaggaggaa gatatgtcct gtggttaata gtgacctaca tagttttaac 3540 agaacaaccc gccgctggtt taccattgag ccagggtgag gtagtgttga tagggaactt 3600 aattactcac acagacattg aggtcgtagt atatttctta ctactctatt tggtcatgag 3660 ggatgagcct ataaagaaat ggatactgct gctattccat gctatgacta acaatccagt 3720 caagactata acagtggcat tgctcatggt tagcggggtt gccaggggtg gaaagataga 3780 tggcggttgg cggcggctgc cggagaccag ctttgacatc caactcgcgc tgacagttat 3840 agtagtcgct gtgatgttgc tagcaaagag agatccgact actgtcccct tggttataac 3900 ggtggcaacc ctgagaacgg ctaagatgac taatggactt agtacggata tagccatagc 3960 tacagtgtca acagcgttgc taacctggac ctacattagt gactattata gatacaagac 4020 ttggctacag taccttatta gcacagtgac aggtatcttt ttaataaggg tactgaaggg 4080 aataggtgag ttggatttac acactccaac cttgccatct tatagacccc tcttcttcat 4140 tctcgtgtac ctcatttcca ctgcagtggt aacaagatgg aatctggaca tagccggatt 4200 gctgttgcag tgtgtcccaa ccctcttgat ggtttttacg atgtgggcag atattctcac 4260 cttgatcctc atactgccca cttacgagtt aacaaagcta tattacctca aggaagtgaa 4320 gattggggca gaaaggggct ggttatggaa gaccaacttc aagagggtaa acgacatata 4380 cgaggtagac caagctggtg aaggggtata ccttttcccg tcaaaacaaa aaacaagttc 4440 aataacaggt accatgttgc cattgattaa agccatactc atcagctgca tcagtaataa 4500 gtggcagttc atatacctat tgtacttgat atttgaagtg tcttactacc tccacaagaa 4560 gatcatagat gaaatagcag gagggaccaa cttcatctca agacttgtag ccgctttgat 4620 cgaagccaat tgggcctttg acaacgaaga agttagaggt ttaaagaagt tcttcctgtt 4680 gtctagtagg gttaaagaac tgatcatcaa acacaaagtg aggaatgaag taatggtcca 4740 ctggtttggt gacgaagagg tttatgggat gccgaagttg gttggcttag tcaaggcagc 4800 aacattgagt aaaaataaac attgtatttt gtgcaccgtc tgtgaagaca gagagtggag 4860 aggagaaacc tgcccaaaat gcgggcgttt tgggccacca atgacctgtg gaatgaccct 4920 tgccgacttt gaagaaaaac actataagag gatctttttt agagaggatc aatcagaagg 4980 gccggttaga gaggagtacg cagggtatct gcaatacaga gccagagggc aattattcct 5040 gaggaatctc ccagtgctag caacaaaagt caagatgctc ctggtcggaa atcttgggac 5100 ggaggtggga gatttggaac accttggctg ggttcttaga gggcctgccg tttgcaagaa 5160 ggttaccgaa catgagaaat gcaccacatc cataatggat gaattgactg cttttttcgg 5220 tgttatgcca aggggcacca cacctagagc ccctgtgaga ttccccacct ctctcttaaa 5280 gataagaagg gggttagaaa ctggctgggc gtacacacac caaggtggca ttagttcagt 5340 ggaccatgtc acttgcggga aagacttact ggtatgtgac actatgggcc ggacaagggt 5400 cgtttgccaa tcaaataata agatgacaga cgagtgcgag tatggagtta aaactgactc 5460 cggatgcccg gaaggagcta ggtgttatgt gttcaaccca gaggcagtta acatatcagg 5520 gactaaagga gccatggtcc acttacaaaa gactggagga gaattcacct gtgtgacagc 5580 atcaggaact ccggccttct ttgatctcaa gaacctcaaa ggctggtcag ggctaccgat 5640 atttgaggca tcaagtggaa gggtagtcgg cagggtcaag gtcgggaaga atgaggactc 5700 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> 735 <212> DNA <213> Artificial Sequence <220> <223> Bovine Viral Disease Virus gene sequence <400> 2 gaaaaagcat tgctggcatg ggcaataata actatagttt tgtttcaagt tacaatggga 60 gaaaacataa cacagtggaa cctacaagac aatgggacgg aagggataca acgggcaatg 120 tttcaaaggg gtgtgaatag aagtctacat ggaatctggc cagagaaaat ctgtactggt 180 gtcccttccc atctagccac cgatatggaa ctaaaaacaa tccatggtat gatggatgca 240 agtgagaaga ccaactacac gtgttgcaga cttcaacgcc atgagtggaa caagcatggt 300 tggtgcaact ggtacaatat tgaaccctgg attctagtca tgaatagaac ccaagccaat 360 cttactgagg gacaaccacc aagggagtgc gcagtcactt gcaggtatga tagggatagt 420 gacttaaacg tagtaacaca agctagagat agccccacac tcttgacagg ttgcaagaaa 480 ggaaagaact tctcctttgc aggcatactg acgcggggtc cctgcaactt tgaaatagct 540 gcgagtgatg tattattcaa agaacatgac tgcactagta tgttccagga tactgctcat 600 taccttgttg acgggatgac caactcctta gaaaatgcca gacaaggaac cgctaaactg 660 acaacctggt taggcaagca gctcgggata ctagggaaaa agttggaaaa caagagtaag 720 acgtggtttg gagca 735 <210> 3 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> forward primer for rt-PCR of Flc-LOM-BErns virus <400> 3 ggaagaaatt agaaaaagca ttgctggcat gggcaat 37 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for rt-PCR of Flc-LOM-BErns virus <400> 4 gatagggcat atgctccaaa ccacgtctta ctc 33  

Claims (6)

Swine fever vaccine virus Flc-LOM-BErns virus (KFCC11442P) having the nucleotide sequence of SEQ ID NO: 1. According to claim 1, wherein the vaccine virus is swine fever vaccine virus Flc-LOM-BErns virus characterized in that by replacing the Erns gene of CSFV (Classical Swine Fever Virus) with the Erns gene of Bovine Viral Disease Virus (BVDV). The swine fever vaccine virus Flc-LOM-BErns virus according to claim 1, wherein the vaccine virus is any one or more markers selected from among Erns genetic markers, Erns serological markers, and END biomarkers. The live swine fever vaccine comprising the swine fever vaccine virus Flc-LOM-BErns virus (KFCC11442P) 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; And Removing the Erns gene from the synthesized cDNA, and inserting the Erns gene of BVDV to prepare a cDNA including BVDV Erns gene; swine fever vaccine virus Flc-LOM-BErns virus (KFCC11442P) Manufacturing method. The method according to claim 5, wherein the prepared cDNA was cloned into a plasmid to produce a Flc-LOM-BErns virus recombinant plasmid, transcribed in vitro to prepare RNA, and then mixed lipofectin (lipofectin) and porcine kidney cells. Method of producing a swine fever vaccine virus Flc-LOM-BErns virus, characterized in that the step of transfecting further comprises the step of producing the Flc-LOM-BErns virus.

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