KR20110131733A - Recombinant swine influenza a virus comprising the gene of a/korea/cj01/09 and a/swine/korea/can01/04, and the swine influenza a virus vaccine comprising the same - Google Patents

Abstract

PURPOSE: A recombinant swine H1N1 virus and a virus vaccine containing the same are provided to protect domestic piggery from the virus and to suppress mutant virus expression. CONSTITUTION: A recombinant swine H1N1 virus contains a gene of sequence numbers 1-6 in an internal gene of A/swine/Korea/CAN01/04 (H1N1) virus of deposit number KCTC 11165BP and virus surface protein gene of deposit number KCTC 11690BP. The virus surface protein gene has HA of sequence number 7 and NA gene of sequence number 8. A vaccine for swine H1N1 virus contains the recombinant swine H1N1 virus.

Description

Recombinant swine influenza A virus comprising the genes of A / Ｋｏｒｅａ / ＣＪ01 / 09 and A / ｓｗｉｎｅ / Ｋｏｒｅａ ／ ＣＡＮ01 / 04, and the swine influenza virus vaccine comprising the same. CJ01 / 09 and A / swine / Korea / CAN01 / 04, and the swine influenza A virus vaccine comprising the same}

The present invention relates to a recombinant swine influenza A virus and a swine influenza A virus vaccine comprising the same. More specifically, the swine influenza A virus is used as a backbone and the domestic human influenza virus A / Korea / CJ01 It relates to a recombinant swine influenza A virus prepared by including the HA and NA genes of / 09 and swine swine influenza A virus vaccine comprising the same.

Swine flu A (H1N1) virus is a respiratory disease that occurs in pigs and is the cause of respiratory diseases that infect humans worldwide. It is called Swine Influenza (SI) or Swine Flu. have. In recent years, swine flu infection has been reported in many countries, including the United States, Canada, Japan, and Australia, and has been reported in domestic pig farms since December 2009.

Pigs, on the other hand, are also widely known as mixing vessels (mixing vessels) that produce new recombinant viruses by mixed infection of human, avian and swine viruses.

Therefore, in the present invention, the gene of the swine influenza A virus isolated from swine is used as a backbone, and when the recombinant swine influenza A virus is prepared by including the surface protein of the new influenza A virus isolated from domestic humans, It has been found that the appearance can be prevented and the present invention has been completed.

Therefore, an object of the present invention is a swine influenza A (H1N1) virus that can suppress the infection and virus discharge of pigs when swine influenza A (H1N1) virus in the pig farm and suppress the emergence of new recombinant influenza virus in pigs and this It is to provide a vaccine comprising.

In order to achieve the above object, the present invention includes the genes of SEQ ID NOs: 1 to 6 and the surface protein genes of Accession No. KCTC 11690BP among the internal genes of A / swine / Korea / CAN01 / 04 (H1N1) virus of Accession No. KCTC 11165BP. To provide a recombinant swine influenza A virus.

In another aspect, the present invention provides a swine influenza A virus vaccine comprising the recombinant swine influenza A virus.

After the immunization of mice and mini-pig with the recombinant virus vaccine according to the present invention, the efficacy of the swine influenza A (H1N1) virus was evaluated. As a result, the protective effect and growth inhibition against swine influenza A (H1N1) virus were evaluated. The ability was confirmed to be excellent.

Therefore, if the recombinant swine influenza A virus according to the present invention is utilized, not only can domestic pig farms be protected from swine influenza A (H1N1) virus, but also prevent the emergence of a variant virus that can appear in pigs.

1 is a cleavage map of the recombinant swine influenza A virus according to the present invention.
Figure 2 is a schematic diagram showing the process of producing a recombinant swine influenza A virus CL A / Korea / CJ01 / 09 x CAN01 using a classical method.
Figure 3 is a schematic diagram showing a process for producing a recombinant swine influenza A virus CL A / Korea / CJ01 / 09 x CAN01 using Reverse Genetics.

The present invention was prepared by using the internal genes of SEQ ID NOs: 1 to 6 of the swine influenza A (H1N1) virus isolated from swine as the backbone, and including the surface protein of the new influenza A (H1N1) virus isolated from domestic humans. It relates to a recombinant swine influenza A virus.

The swine influenza A (H1N1) virus used in the present invention is A / swine / Korea / CAN01 / 04 (H1N1) of Accession No. KCTC 11165BP, more specifically, the A / swine / Korea / CAN01 / 04 (H1N1) virus. The genes of SEQ ID NOS: 1 to 6 encoding the M, NP, NS, PA, PB1, and PB2 proteins are used in the internal genes.

In addition, the new human influenza A (H1N1) virus isolated from the present invention was named as "A / Korea / CJ01 / 09 (H1N1)", which was assigned to the Korean Collection for Type Cultures, an international depository institution. The deposit was made on April 27, 2010, and was assigned accession number KCTC 11690BP. In the present invention, the genes of SEQ ID NOs: 7 and 8 encoding HA and NA proteins, which are surface proteins of the virus, are used. The HA and NA genes show 99.9% homology with the genes of the recent pandemic A / H1N1 / 2009 virus, and are relatively different from the conventional swine virus with about 90% homology.

As shown in FIG. 1, the recombinant swine influenza A virus according to the present invention has the M, NP, NS, PA, PB1 and PB2 genes of SEQ ID NOs: 1 to 6, and includes the HA and the sequence of SEQ ID NO: 7 It comprises the NA gene of No. 8 (CL A / Korea / CJ01 / 09 x CAN01).

The recombinant swine flu influenza A virus according to the present invention is not particularly limited in its preparation method, and can be easily prepared by those skilled in the art by conventional methods well known in the art. In one embodiment of the present invention, recombinant swine influenza A virus CL A / Korea / CJ01 using an intracellular (fertilized egg) complex infection and plaque assay, which is a classical recombinant virus manufacturing method, as shown in FIG. 2. Only / 09 x CAN01 can be purely separated. In another embodiment of the present invention, as shown in FIG. 3, recombinant swine influenza A virus may be prepared in vitro through reverse genetics, but the present invention is not limited thereto.

The swine influenza A virus vaccine according to the present invention may use the recombinant swine influenza A virus itself, or may be used with additives, adjuvants, or carriers that are commonly used for the recombinant swine influenza A virus.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. These examples are provided only for understanding the contents of the present invention, and the scope of the present invention is not limited to these examples, and modifications, substitutions, and insertions commonly known in the art may be performed. This is also included in the scope of the present invention.

Example 1 A / Korea / CJ01 / 09 (H1N1) Virus Isolation

The nasopharyngeal secretions collected from a patient hospitalized with swine flu at Chungbuk National University Hospital were placed in a cell culture medium containing antibiotics, a turbidity solution was prepared, and centrifuged to remove the secretions. After infection, the virus was isolated by incubating in a CO ₂ cell culture for 48 hours. The isolated virus was confirmed to be a new influenza virus through RT-PCR and genetic analysis.

The new human influenza virus isolated from above was named “A / Korea / CJ01 / 09 (H1N1)”, and it was issued to the Korean Collection for Type Cultures, an international depository institution, on April 27, 2010. Was deposited with the accession number KCTC 11690 BP.

Example 2 Preparation of Recombinant Swine Influenza A Virus (1)

A / Korea / CJ01 / 09 (H1N1) virus isolated in Example 1 and A / swine / Korea / CAN01 / 04 (H1N1) isolated from domestic pigs were simultaneously infected with MDCK cells and then A / swine / Korea / An antibody against the CAN01 / 04 virus was added.

When the cytopathic effect (CPE) is observed, the supernatant of the culture supernatant was preliminarily stepped on MDCK cells (pre-cultured in 6-well plates), and then infected and subjected to the Prag test. The cells were covered with agar (Agarose) 0.7% medium mixture and incubated for 48 hours in a 37 ℃ CO ₂ incubator. After 48 hours, plaques were observed and the plaques were collected and inoculated into 10-day-old fertilized eggs.

After 48 hours, genetic analysis of the virus isolated from eggs was carried out. A / swine / Korea / CAN01 / 04 virus containing 6 internal genes and isolated from domestic human strain A / Korea / CJ01 / 09 The recombinant swine influenza A virus containing the HA and NA genes, which are the surface proteins of 09 virus, was selected and the virus thus produced was named CL A / Korea / CJ01 / 09 x CAN01. The virus was incubated in fertilized eggs for vaccine preparation, and then inactivated by treatment with 0.025% formalin containing allantoic fluid.

Example 3 Preparation of Recombinant Swine Influenza A Virus (2)

The HA (SEQ ID NO: 7) and NA (SEQ ID NO: 8) genes of A / Korea / CJ01 / 09 (H1N1) isolated in Example 1 were inserted into the expression vector pHW2000, respectively. Meanwhile, M (SEQ ID NO: 1), NP (SEQ ID NO: 2), NS (SEQ ID NO: 3), PA (SEQ ID NO: 4), PB1 (SEQ ID NO: 5), and PB2 (SEQ ID NO: A / Swine / Korea / CAN01 / 04) 6) The genes were prepared by inserting them into the expression vector pHW2000. The expression vectors into which the genes of SEQ ID NOS: 1 to 8 were inserted were mixed with the transfection reagent (Mirus), and left at room temperature for 40 minutes.

The vector mixture was carefully added to Vero cells prepared 24 hours before, and after 6 hours, the culture was replaced with serum-free culture. After 36 hours after transfection, the culture solution containing TPCK-trypsin was added and supernatants were inoculated using 10-day-old fertilized eggs from 48 hours later.

After 48 hours, the recombinant swine influenza A virus isolated from the eggs was inactivated in the same manner as in Example 2.

Test Example 1 Antigenicity Evaluation through Animal Experiment

<Mouse model>

In order to evaluate the antigenicity of the recombinant vaccine candidate virus CL A / Korea / CJ01 / 09 x CAN01 developed in the present invention, the recombinant virus prepared in Example 3 and A / swine / which is a swine influenza virus isolated from domestic pigs Korea / CAN01 / 04 isolates were inoculated with 10 10-day-old fertilized eggs each 10 hours later, and after 48 hours, urine was detected and titer was confirmed by a Hemagglutinin test with 0.5% turkey RBC.

The isolated urethra was treated with formalin at a concentration of 0.025%, stored at 4 ° C. for 72 hours, and then inoculated into 10-day-old fertilized eggs to show that there was no virus that was completely inactivated.

Inactivated virus urea was placed in a SW28 swing rotor bucket (Beckman, USA) with 20% sucrose cushion and settled at 25,000 rpm for 3 hours to purely isolate virus. After measuring the protein amount of the isolated virus was made to the HA protein 3.5 ㎍ / 100 ㎕.

Antigenity was assessed by mixing 2% alum with the desired concentration of virus solution and intramuscularly inoculating mice (3.5 μg / 100 μl; primary inoculation) 24 hours later. Two weeks after the virus inoculation, the second inoculation was performed in the same manner as above.

In order to confirm the presence of antibody after vaccination with oral virus, sera were isolated from mouse orbits and treated with RDE, and the swine flu virus A / Korea / CJ01 / 09 and the swine flu virus A / swine / Korea / CAN01 / Hemagglutinin inhibition (HI) test was performed using 04 virus, and the results are shown in Table 1 below.

Inoculation Virus Vaccine prescription Antibody changes HA content (3.5 ㎍) A / swine / Korea / CAN01 / 04 A / Korea / CJ01 / 09 A / swine / Korea / CAN01 / 04 1st inoculation 20-40 <20 2nd inoculation 80-320 20 CL A / Korea / CJ01 / 09 x CAN01 1st inoculation <20 40-80 2nd inoculation 20-40 160-320

In the results of Table 1, the serum isolated two weeks after the first virus inoculation did not react with the heterologous virus at all, and two weeks after the second inoculation, the titer of 20-40 with the heterologous virus was It was judged that the antigenicity was low. However, homologous viruses were found to have high antigenicity, with very high antibody titers (160-320) in the sera of mice inoculated with two vaccine candidate viruses two weeks after the second inoculation.

<Mini pig model>

Prepared by inactivating CL A / Korea / CJ01 / 09 x CAN01 virus and swine flu virus A / swine / Korea / CAN01 / 04 prepared in Example 3 in the same manner as in the mouse model, and inoculated once per animal It was made to the amount of HA protein of 7.5 ㎍ / ml, mixed with 2% alum and stored at 4 ℃ for 24 hours.

The prepared vaccine was injected into the muscle 1 ml per horse, and after 2 weeks, serum was collected and a second inoculation was performed. The isolated serum was subjected to hemagglutination inhibition test in the same manner as in the mouse experiment, the results are shown in Table 2 below.

Inoculation Virus Vaccine prescription Antibody changes HA content (7.5 ㎍) A / swine / Korea / CAN01 / 04 A / Korea / CJ01 / 09 A / swine / Korea / CAN01 / 04 1st inoculation 40-80 <20 2nd inoculation 160-320 20 CL A / Korea / CJ01 / 09 x CAN01 1st inoculation <20 40-80 2nd inoculation 20-40 320

In the results of Table 2, the serum collected two weeks after the first vaccination showed 40-80 antibody titers in the homologous test for each virus, whereas in the heterologous test, As with the results, no cross reactions occurred.

In addition, the result of 2 weeks after the second vaccination also had a cross-reactivity of about 20-40 in the heterogeneous test similarly to the mouse result, but in the homogeneous test, it showed high antibody titer (320) and high immunogenicity.

[Test Example 2] Defense ability by attack vaccination

In order to confirm the protective ability by inoculating recombinant swine influenza A virus according to the present invention, i) swine influenza A / swine / Korea / CAN01 / 04 virus, or ii) CL A / Korea / CJ01 / Two weeks after 09 x CAN01 virus inoculation at 2 week intervals, two weeks later, swine influenza virus A / Korea / CJ01 / 09 and swine influenza A / swine / Korea / CAN01 / 04 virus 10 ⁵ TCID ₅₀ / ml respectively. Inoculation. After 2, 4, 5, 6, 7 and 8 days of challenge, non-juice samples were taken to check whether the virus was discharged, and the results are shown in Table 3 below. In addition, the presence of airborne infection was confirmed by including the cohabitation group, virus titer was confirmed by inoculation (log ₁₀ EID ₅₀ / ml), and the detection sensitivity was 0.7log ₁₀ EID ₅₀ / ml (“-” is the virus). Is not detected and “*” is the standard deviation).

Work
(day) Virus titer (log ₁₀ EID ₅₀ / ml) A / Korea / CJ01 / 09 Challenge A / swine / Korea / CAN01 / 04 Challenge A / swine / Korea / CAN01 / 04 CL A / Korea / CJ01 / 09 x CAN01 A / swine / Korea / CAN01 / 04 CL A / Korea / CJ01 / 09 x CAN01 infection Air propagation infection Air propagation infection Air propagation infection Air propagation -One - - - - - - - - 0 - - - - - - - - 2 5 (0.3) * - 2.5 (0.3) - 4.7 (0.3) - 2.3 (0.3) - 4 5 (0.5) 2 (0.3) 1.7 (0.2) - 5 (0.5) 2 (0.3) 1.7 (0.2) - 5 3 (0.3) 2 (0.3) - - 2.3 (0.3) 2.5 (0.3) - - 6 - 1 (0.3) - - - 1 (0.3) - - 7 - - - - - - - - 8 - - - - - - - -

In the results of Table 3, the pigs vaccinated by inactivating the recombinant swine influenza A virus prepared in Example 3 made short virus release and did not occur by air propagation against swine flu challenge.

However, pigs immunized with swine influenza A / swine / Korea / CAN01 / 04 confirmed that the virus was released for a longer period of influenza vaccination and that infection with domestic pigs was also carried out through air transmission. In other words, even when immunized with a homologous virus when inoculated with the swine influenza A / swine / Korea / CAN01 / 04 swine influenza virus inactivated recombinant swine influenza A virus according to the present invention was protected. In case of immunization with recombinant swine influenza A virus, it was confirmed that there is no protection.

Korea Research Institute of Bioscience and Biotechnology KCTC11690BP 20100427

<110> REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries.National Veterinary Research and Quarantine Service (NVRQS)) <120> Recombinant swine influenza A virus comprising the gene of          A / Korea / CJ01 / 09 and A / swine / Korea / CAN01 / 04, and the swine          influenza A virus vaccine comprises the same <130> YPD / 201004-0025 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 1108 <212> DNA <213> Swine / Korea / CAN01 / 04, M <400> 1 gcagttaacc ggagtactgg tcgacctccg aagttggggg ggagcaaaag caggtagatg 60 tttaaagatg agtcttctaa ccgaggtcga aacgtatgtt ctctctatcg tcccgtcagg 120 ccccctcaaa gccgaaatag cacagagact cgaagacgtt tttgcaggga aaaacaccga 180 tcttgaggct ctcatggaat ggctaaagac aagaccaatc ctgtcacctc tgactaaggg 240 gattttaggg tttgtgttca cgctcaccgt gcccagtgag cgaggactgc agcgtagacg 300 ttttgtccag aatgccctca atgggaatgg tgacccgaac aacatggaca aggcggtcaa 360 actgtacagg aaactaaaaa gggaaataac attccatggg gccaaagaag tagcgctcag 420 ttactctgct ggtgcacttg ccagttgcat gggcctcata tacaacagaa tggggactgt 480 caccactgag gtggcctttg gtctggtatg cgcaacctgt gaacagattg ctgattctca 540 gcatcgatct catagacaaa tggtgacaac aaccaatcca ctaatcaggc acgagaacag 600 aatggtaata gccagcacaa cagctaaagc catggaacaa atggctggat caagcgaaca 660 agcagcagag gctatggagg ttgccagcca ggctagacaa atggtacagg caatgagaat 720 aattgggact caccctagtt ccagcactgg tctaaaagat gatcttcttg aaaatttaca 780 ggcctatcag aagcggatgg gagtgcaaat gcaacgattc aagtgatcct ctcatttatg 840 ccgcaagcat cattgggatt ttgcacctga tattgtggat tcttgatcgt ctttttttca 900 aatgcattta ccgtcgcttt aaatacggtc tgcaaagagg gccttctacg gaaggagtgc 960 cggagtccat gagggaagaa tatcgacaga aacagcagag tgctgtggat gttgacgatg 1020 gtcattttgt caacatagtg ctagagtaaa aaactacctt gtttctacta ataacccggc 1080 ggcccaaaat gccgactcgg agcgaaag 1108 <210> 2 <211> 1723 <212> DNA <213> Swine / Korea / CAN01 / 04, NP <400> 2 cggggggccg cggcgtggtt cggtgagcga cctcccggcc ccgggggagg tatatctttc 60 gctccgagtc ggcattttgg gccgccgggt tattagtaga aacaagggta tttttcctca 120 actgtcatac tcctctgcat tgtctccgaa gaaataagat ccttcattac tcatgtcaaa 180 ggaaggcacg atcgggctcg ttgccttttc gtccgagagc tcgaagactc cccgcccctg 240 gaaggacaaa tcttctggct ttgcactttc catcatcctt ataacttctg ttcgcatatc 300 ggatgtccgt ccttcattgt tcccgctgaa agctgccata acggttgctc tttcaaaagg 360 gagattccgt tgcactgaga atgtaggttg cacactgatc tggcccgcgg atgctttctg 420 ttgattggta tttcctccac ttctggtcct tatggcccag tatctgcttc tcagctctaa 480 ggtattggaa tccatggctt ccacattctc atttgaagca atctgaaccc ctcttgtgga 540 aagctttcct cttggaatca ctttcttccc tcttatgaaa cttgataccc ttagatcctc 600 aaatgcagca gagtggcatg ccacccacac caattgactc ttgtgagctg ggttttcatt 660 tggtcttatc aggctgaaca cttgactgtt ttggagtaat ttaaatgggt ctatcccgac 720 cagtgaatac ccttcccttt caaagtcatg cccacttgcc actgcaagcc catacacaca 780 agcaggcagg caagacttat gtgcaactga tccccttaga ataagtgctg accgtgccag 840 gaaaatgaga tcttcaatct cagcgtttcc tgggttccga ctttctctca cttgatccat 900 cattgccctc tgggcagctg tctgaaattt tcctttgaga atattgcaca ttctctcata 960 tgcaatcctt gtccttcgtc cattttcacc tctccagaag tttcggtcat tgaccccacg 1020 tttgatcatt ctgattaatt ccattgccat tgttccaact cctttcactg cagcacctgc 1080 ggccccagac cttctgggaa gtgttgaacc ttgcattaga gagcacattc tgggatccat 1140 tccagtgcga acaagcgctc ttgttctctg gtacgtggca tcattcagat tggaatgcca 1200 aatcatgata tgagtaagac cagctgttgc atcttcacca ttgtttgctt ggcgccaaac 1260 tctccttatt tcttctttgt cataaagaat gagttctctc atccactttc cgtttactct 1320 tctatatatg ggtcctccag ttttcttagg atccttccca gcactgggat gctcttctag 1380 gtatttattt cttctctcat cgaaagcaga gagcaccatt ctctctattg ttatgctatt 1440 ttggattagt cgtccctcat agtcactgag tttgagttca gtgcacattt ggatgtagaa 1500 tttcccgatt ccaccaatca ttcttccgac agatgctctg atttctgtgg catcctggcg 1560 ttccccacca gtctccattt gttcatatga tcgtttggtg ccttgagacg ccatggcttc 1620 gatgtcactc attgagtgat tatctaccct gcttttgctc ccccccaact tcggaggtcg 1680 accagtactc cggttaactg tagcgtaaac cgggttaaat ctc 1723 <210> 3 <211> 1193 <212> DNA <213> Swine / Korea / CAN01 / 04, NS <400> 3 gcctgtttaa cgctagcagt taaccggagt actggtcgac ctccgaagtt gggggggagc 60 aaaagcaggg tgacaaagac ataatggact ccaatactgt gtcaagcttt caggtagact 120 gtttcctttg gcacatccgc aaacgatttg cagacaatgg attgggtgat gccccattcc 180 ttgatcggct ccgccgagat caaaagtccc taaaaggaag aggcaacacc cttggcctcg 240 acatcgaaac agccactctt gttgggaaac aaattgtgga gtggattttg aaagaggaat 300 ccagcgagac tcttaagatg accattgcat ctgtacctac ttcgcgctac ctagctgaca 360 tgaccctcga ggaaatgtca cgagactggt tcatgctcat gcctaggcaa aagataatag 420 gccctctttg tgtgcgaatg gaccaggcga tcatggaaaa gaacatcata ctgaaagcga 480 acttcagtgt gatctttaac cgattagaga ctttgatact actaagggct ttcactgagg 540 agggagcaat cgttggagaa atttcaccat taccttctct tccaggacat actaataagg 600 atgtcaaaaa tgcagttggg gtcctcatcg gaggacttga atggaatggt aacacggttc 660 gaggctctga aaatctacag agattcactt ggagaaaccg taatgaggat gggagacctt 720 cactacctcc agagcagaaa tgaaaagtgg cgagagcaat tgggacagaa atttgaggaa 780 ataaggtggt taattgaaga agtgcggcac agattgaaag cgacagagaa tagtttcgaa 840 caaataacat ttatgcaagc cttacaacta ctgcttgaag tagaacaaga gataaggact 900 ttctcgtttc agcttattta atgataaaaa acacccttgt ttctactaat aacccggcgg 960 cccaaaatgc cgactcggag cgaaagatat acctcccccg gggccgggag gtcgcgtcac 1020 cgaccacgcc gccggcccag gcgacgcgcg acacggacac ctgtccccaa aaacgccacc 1080 atcgcagcca cacacggagc gcccggggcc ctctggtcaa ccccaggaca cacgcgggag 1140 cagcgccggg ccggggacgc cctcccggcg gtcacctaaa tgctagagct cgc 1193 <210> 4 <211> 2242 <212> DNA <213> Swine / Korea / CAN01 / 04, PA <400> 4 ggggggagcg aaagcaggta ctgatccaaa atggaagact ttgtgcgaca atgcttcaat 60 ccaatgattg tcgagcttgc ggaaaaggca atgaaagaat atggagaaga tccgaaaatc 120 gaaactaaca aattcgctgc aatatgcaca cacttggaag tctgtttcat gtattcggat 180 ttccatttca tcgacgaacg gggtgaatca atcattgtag aatctggtga tccaaatgca 240 ttactgaagc accggtttga gataattgaa ggaagagacc ggaccatggc ctggacagtg 300 gtgaacagta tctgcaacac cacaggggta gagaagccta aatttcttcc ggatttatac 360 gattacaaag aaaaccgatt cattgaaatt ggggtgacac gaagggaggt ccacatatac 420 tacctagaaa aagccaacaa aataaaatcc gagaagacac acattcacat tttttcattc 480 actggagagg agatggccac caaagcagac tacacccttg atgaagagag cagggcaaga 540 atcaaaacca ggcttttcac cataagacaa gagatggcca gtagaggtct atgggattcc 600 tttcgtcagt ccgagagagg cgaagagaca attgaagaaa gatttgaaat tacaggaacc 660 atgcgcaggc ttgcagacca aagtctccca ccgaacttct ccagccttga aaactttaga 720 gcctatgtag atgggttcga accaaacggc tgcattgagg gcaagctttc tcaaatgtca 780 aaagaagtga gcgcccaaat tgaaccattc ttgaagaaaa caccacgccc tctcagatta 840 cctgatgggc ctccttgctc tcagcggtca aagttcttgc tgatggatgc tctgaaacta 900 agtattgaag acccgagtca tgagggggaa ggaataccac tatatgatgc aatcaagtgc 960 atgaagacat ttttgggctg gaaagagcct aacataatca aaccacatga gaaaggcata 1020 aaccccaatt acctactagc ttggaagcag gtgctagcag agctccagga cattgaaaat 1080 gaagagaaga tcccaaagac aaagaacatg aagagaacaa gccaattgaa gtgggcactc 1140 ggtgagaata tggcaccaga aaaagtagac tttgatgact gcaaagatgt tggtgacctt 1200 aaacagtatg acagcgatga gccagagccc aaatctctag caagctgggt ccaaaatgaa 1260 ttcaataagg catgtgaatt gaccgattca agctggatag aacttgatga gataggagaa 1320 gatgttgccc cgattgaaca catagcaagc atgaggagga actattttac agcagaagtg 1380 tcccattgca gggctactga atacataatg aagggagtgt acataaatac ggctttgctt 1440 aatgcatctt gtgcagccat ggatgacttt cagctgatcc caatgataag caaatgtagg 1500 accaaagaag gaagacgaaa gacaaatctg tatgggttta ttataaaagg aaggtcccat 1560 ctgagaaatg atactgacgt ggtgaacttt gtaagtatgg agttctccct cactgacccg 1620 agactggagc cacacaaatg ggaaaaatac tgtgttcttg aaataggaga catgctcctg 1680 aggactgcga taggccaagt gtcgaggccc atgttcctat atgtgagaac caatggaacc 1740 tccaagatca agatgaaatg gggcatggaa atgaggcgct gccttcttca gtctcttcag 1800 cagattgaga gcatgattga ggctgagtct tctgtcaaag agaaagacag gaccaaggaa 1860 ttctttgaaa acaaatcgga aacatggcca atcggagaat cacccaaagg agtggaggaa 1920 ggctctattg ggaaagtgtg caggacctta ctggcaaaat ctgtattcaa cagtctatac 1980 gcgtctccac aacttgaggg gttttcggct gaatcgagaa aattgcttct cattgttcag 2040 gcacttaggg acaacctgga acctggaacc ttcgatcttg gagggctata tgaagcaatc 2100 gaggagtgcc tgattaatga tccctgggtt ttgcttaatg cgtcttggtt caactccttc 2160 ctcacacatg cactgaaata gttgtggcaa tgctactatt tgctatccat actgtccaaa 2220 aaagtacctt gtttctacta at 2242 <210> 5 <211> 2349 <212> DNA <213> Swine / Korea / CAN01 / 04, PB1 <400> 5 gggagcgaaa gcaggcaaac catttgaatg gatgtcaatc cgactctact ttttctaaag 60 gttccagcgc aaaatgccat aagcaccaca ttcccttata ctggagatcc tccatacagc 120 catggaacag gaacaggata caccatggac acagtcaaca gaacacacca atattcagaa 180 aaagggaagt ggacgacaaa cacagagact ggggcacccc agctcaaccc gattgatgga 240 ccactacctg aggataatga accaagtgga tatgcacaaa cagactgtgt tctggaggcc 300 atggctttcc ttgaagaatc ccacccaggg atatttgaga attcatgcct tgaaacaatg 360 gaagttgttc aacaaacaag ggtggataaa ctaactcaag gtcgccagac ttatgattgg 420 acattaaaca gaaatcaacc ggcagcaact gcattggcca acaccataga agtttttaga 480 tcgaatggtc taacagctaa tgagtcagga aggctaatag atttcctaaa ggatgtgatg 540 gaatcaatgg ataaagagga aatagagata acaacacact ttcaaagaaa aaggagagta 600 agagacaaca tgaccaagaa gatggtcaca caaagaacaa taggaaagaa aaaacaaaga 660 gtgaataaga gaggttatct aataagagca ctgacattga atacgatgac caaagatgca 720 gagagaggca aattaaaaag aagggctatc gcaacacctg ggatgcaaat tagagggttc 780 gtgtactttg ttgaaacttt agctaggagc atttgcgaaa agcttgaaca gtctggactc 840 ccagtagggg gcaatgaaaa gaaagccaaa ttggcaaatg ttgtgagaaa gatgatgact 900 aattcacaag acacagagct ttctttcaca atcactgggg acaacactaa gtggaatgaa 960 aatcaaaatc ctcgaatgtt cctggcgatg attacatata tcaccagaaa tcaacccgag 1020 tggttcagaa acatcctgag catggcaccc ataatgttct caaacaaaat ggcaagacta 1080 ggaaaagggt acatgttcga gagtaaaaga atgaagctcc gaacacaaat accagcagaa 1140 atgctagcaa gcattgacct gaagtatttc aatgaatcaa caaggaagaa aattgagaaa 1200 ataaggcctc ttctaataga tggcacagca tcattgagcc ctggaatgat gatgggcatg 1260 ttcaacatgc taagtacggt tttgggagtc tcgatactga atcttggaca aaagaaatac 1320 accaggacaa catactggtg ggatggactc caatcctccg acgattttgc cctcatagtg 1380 aatgcaccaa atcatgaggg aatacaagca ggagtggata gattctacag gacctgcaag 1440 ttagtgggaa tcaacatgag caaaaagaag tcctatataa ataagacagg gacatttgaa 1500 ttcacaagct ttttttatcg ctatggattt gtggctaatt ttagcatgga gctgcccagt 1560 tttggagtgt ctggaataaa tgaatcagct gatatgagta ttggagtaac agtgataaag 1620 aacaacatga taaacaatga ccttggacct gcaacagccc agatggccct tcaattgttc 1680 atcaaagact acagatacac atataggtgc catagaggag acacacaaat tcagacgaga 1740 agatcattcg agctaaagaa gctgtgggat caaacccaat caaaggcagg actattagta 1800 tctgatggag gaccaaactt atacaatatc cggaatcttc acattcctga agtctgctta 1860 aaatgggagc taatggatga ggattatcgg ggaagacttt gtaatcccct gaaccccttt 1920 gtcagccata aagagattga ttctgtaaac aatgctgtgg tgatgccagc ccatggtcca 1980 gccaagagca tggaatatga tgccgttgca actacacact cctggattcc caagaggaac 2040 cgctctattc tcaacacaag ccaaagggga attcttgagg atgaacagat gtaccagaag 2100 tgctgcaacc tgttcgagaa atttttccct agtagttcat acaggagacc ggttggaatt 2160 tctagcatgg tggaggccat ggtgtctagg gcccggattg atgccaggat tgacttcgag 2220 tctggacgga ttaagaaaga agagttctct gagatcatga agatctgttc caccattgaa 2280 gaactcagac ggcaaaaata atgaatttag cttgtccttc atgaaaaaat gccttgtatc 2340 tactaataa 2349 <210> 6 <211> 2385 <212> DNA <213> Swine / Korea / CAN01 / 04, PB2 <400> 6 gggagcgaaa gcaggtcaaa tatattcaat atggagagaa taaaagaact aagagatcta 60 atgtcgcagt cccgcactcg cgagatactc actaagacca ctgtggacca tatggccata 120 atcaaaaagt acacatcagg aaggcaagag aagaaccccg cactcagaat gaagtggatg 180 atggcaatga aatacccaat tacagcagac aagagaataa tggacatgat tccagagagg 240 aatgaacaag gacaaaccct ctggagcaaa acaaacgatg ctggatcaga ccgcgtgatg 300 gtatcacctc tggccgtaac atggtggaat aggaatggcc caacaacaag tacagttcac 360 taccctaagg tatataaaac ttatttcgaa aaagtcgaaa ggttaaaaca tggtaccttt 420 ggccctgtcc acttcagaaa tcaagttaaa ataagaagga gagttgacac aaaccctggt 480 cacgcagatc tcagtgccaa ggaggcacag gatgtgatca tggaagttgt tttcccaaat 540 gaagtggggg caagaatact gacatcagag tcacagctga caataacaaa agagaagaaa 600 gaagagctcc aggattgtaa aattgctccc ttgatggtgg catacatgct agaaagagag 660 ttggtccgta aaacgaggtt tctcccggtg gctggtggaa caggcagtgt ttatattgag 720 gtgctgcact taacccaggg gacatgctgg gagcagatgt acactccagg aggagaagtg 780 agaaatgatg atgttgacca aagtttgatt atcgctgcta gaaacatagt aagaagagca 840 gcagtgtcag cagacccatt agcatctctc ttggaaatgt gccacagcac acagattgga 900 ggaataagga tggtggacat ccttagacag aatccaacgg aggaacaagc cgtagacata 960 tgcaaggcag caatggggtt gaggattagc tcatctttca gctttggtgg gttcactttc 1020 aaaagaacaa gcggatcatc agtcaagaaa gaagaagaag tgctcacggg caacctccaa 1080 acactgaaaa taagagtaca tgaaggatat gaagaattca caatggtcgg gagaagagca 1140 acagctattc tcagaaaggc aaccaggaga ttgatccagt taatagtaag tgggagagac 1200 gagcagtcaa ttgctgaggc aataattgtg gccatggtat tttcacaaga ggattgcatg 1260 atcaaggcag ttaggggcga tctgaacttt gtcaataggg caaaccagcg actgaatccc 1320 atgcaccaac tcttgaggca tttccaaaaa gatgcaaaag tgcttttcca gaactgggga 1380 attgaaccca tcgacaatgt gatgggaatg atcggaatat tgcccgatat gatcccaagc 1440 acggagatgt cgctgagagg gataagagtc agcaaaatgg gagtagatga atactccagc 1500 acggagagag tggtagtgag cattgaccga tttttgaggg ttcgggatca acgagggaac 1560 gtactattgt ctcccgaaga ggtcagcgag acacaaggga cggagaagtt gacaataact 1620 tattcgtcat caatgatgtg ggagatcaat ggtcctgagt cagtgctggt caacacttat 1680 caatggatca tcaggaactg ggaaactgtg aaaattcaat ggtcgcaaga tcccacgatg 1740 ttatacaaca aaatggaatt tgaaccattt cagtctcttg tccctaaggc aaccagaagc 1800 cgatacagtg gattcgtaag gacactgttc cagcaaatgc gggatgtgct tggaacattt 1860 gacactgtcc aaataataaa acttctcccc tttgctgctg ctccaccaga acagagtagg 1920 atgcagtttt cctcattgac tgtgaatgtg agaggatcag ggttgaggat actggtaaga 1980 ggcaattctc cagtattcaa ttacaacaaa gcaaccaaaa ggcttacagt tcttggaaag 2040 gatgcaggtg cattgactga agatccagat gaaggcacag ctggagtgga gtctgctgtc 2100 ctgagaggat ttctcatttt gggcaaagaa gacaagagat atggcccagc attaagcatc 2160 aatgaactga gcaatcttgc aaaaggagag aaggctaatg tgctaattgg gcaaggagac 2220 gtagtgttgg taatgaaacg gaaacgggac tctagcatac ttactgacag ccagacagcg 2280 accaaaagaa ttcggatggc catcaattag tgtcgaattg tttaaaaacg accttgtttc 2340 tactaataac ccggcggccc aaaatgccga ctcggagcga aaaga 2385 <210> 7 <211> 1701 <212> DNA <213> Korea / CJ01 / 09, HA <400> 7 atgaaggcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc agacacatta 60 tgtataggtt atcatgcaaa caattcaaca gacactgtag acacagtact agaaaagaat 120 gtaacagtaa cacactctgt taaccttcta gaagacaagc ataacgggaa actatgcaaa 180 ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga 240 aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacatct 300 agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag 360 caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg 420 cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc 480 ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa 540 tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600 actagtgctg accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggaca 660 tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa 720 gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa 780 gcaactggaa atctagtggt accgagatat gcattcgcaa tggaaagaaa tgctggatct 840 ggtattatca tttcagatac accagtccac gattgcaata caacttgtca gacacccaag 900 ggtgctataa acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt 960 ccaaaatatg taaaaagcac aaaattgaga ctggccacag gattgaggaa tgtcccgtct 1020 attcaatcta gaggcctatt tggggccatt gccggtttca ttgaaggggg gtggacaggg 1080 atggtagatg gatggtacgg ttatcaccat caaaatgagc aggggtcagg atatgcagcc 1140 gacctgaaga gcacacagaa tgccattgac gagattacta acaaagtaaa ttctgttatt 1200 gaaaagatga atacacagtt cacagcagta ggtaaagagt tcaaccatct ggaaaaaaga 1260 atagagaatt taaataaaaa agttgatgat ggtttcctgg acatttggac ttacaatgcc 1320 gaactgttgg ttctattgga aaatgaaaga actttggact accacgattc aaatgtgaag 1380 aacttatatg aaaaggtaag aagccagtta aaaaacaatg ccaaggaaat tggaaacggc 1440 tgctttgaat tttaccacaa atgcgataac acgtgcatgg aaagtgtcaa aaatgggact 1500 tatgactacc caaaatactc agaggaagca aaattaaaca gagaagaaat agatggggta 1560 aagctggaat caacaaggat ttaccagatt ttggcgatct attcaactgt cgccagttca 1620 ttggtactgg tagtctccct gggggcaatc agtttctgga tgtgctctaa tgggtctcta 1680 cagtgtagaa tatgtattta a 1701 <210> 8 <211> 1414 <212> DNA <213> Korea / CJ01 / 09, NA <400> 8 atgaatccaa accaaaagat aataaccatt ggttcggtct gtatgacaat tggaatggct 60 aacttaatat tacaaattgg aaacataatc tcaatatgga ttagccactc aattcaactt 120 gggaatcaaa atcagattga aacatgcaat caaagcgtca ttacttatga aaacaacact 180 tgggtaaatc agacatatgt taacatcagc aacaccaact ttgctgctgg acagtcagtg 240 gtttccgtga aattagcggg caattcctct ctctgccctg ttagtggatg ggctatatac 300 agtaaagaca acagtataag aatcggttcc aagggggatg tgtttgtcat aagggaacca 360 ttcatatcat gctccccctt ggaatgcaga accttcttct tgactcaagg ggccttgcta 420 aatgacaaac attccaatgg aaccattaaa gacaggagcc catatcgaac cctaatgagc 480 tgtcctattg gtgaagttcc ctctccatac aactcaagat ttgagtcagt cgcttggtca 540 gcaagtgctt gtcatgatgg catcaattgg ctaacaattg gaatttctgg cccagacaat 600 ggggcagtgg ctgtgttaaa gtacaacggc ataataacag acactatcaa gagttggaga 660 aacaatatat tgagaacaca agagtctgaa tgtgcatgtg taaatggttc ttgctttact 720 gtaatgaccg atggaccaag tgatggacag gcctcataca agatcttcag aatagaaaag 780 ggaaagatag tcaaatcagt cgaaatgaat gcccctaatt atcactatga ggaatgctcc 840 tgttatcctg attctagtga aatcacatgt gtgtgcaggg ataactggca tggctcgaat 900 cgaccgtggg tgtctttcaa ccagaatctg gaatatcaga taggatacat atgcagtggg 960 attttcggag acaatccacg ccctaatgat aagacaggca gttgtggtcc agtatcatct 1020 aatggagcaa atggagtaaa aggattttca ttcaaatacg gcaatggtgt ttggataggg 1080 agaactaaaa gcattagttc aagaaacggt tttgagatga tttgggatcc gaacggatgg 1140 actgggacag acaataactt ctcaataaag caagatatcg taggaataaa tgagtggtca 1200 ggatatagcg ggagttttgt tcagcatcca gaactaacag ggctggattg tataagacct 1260 tgcttctggg ttgaactaat cagagggcga cccaaagaga acacaatctg gactagcggg 1320 agcagcatat ccttttgtgg tgtaaacagt gacactgtgg gttggtcttg gccagacggt 1380 gctgagttgc catttaccat tgacaagtaa tttg 1414

Claims (4)

The genes of SEQ ID NOs: 1 to 6 among the internal genes of A / swine / Korea / CAN01 / 04 (H1N1) virus of Accession No. KCTC 11165BP and
Recombinant swine influenza A virus comprising the virus surface protein gene of accession number KCTC 11690BP. The recombinant swine influenza A virus according to claim 1, wherein the virus surface protein gene of Accession No. KCTC 11690BP is composed of HA of SEQ ID NO. 7 and NA gene of SEQ ID NO. 8. A swine influenza virus vaccine comprising the recombinant swine influenza A virus according to claim 1. A method of preventing the onset of swine influenza A virus by inoculating a swine influenza virus vaccine according to claim 3 to a pig.

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