KR20140010582A - Recombinant expression vector for preparing highly productive and avirulent influenza virus, and use thereof - Google Patents

Abstract

The present invention relates to: a composition for high productiveness of an embryonated egg of H1N1 family avian influenza and non-pathogenicity of a mouse including, as a active ingredient, a polynucleotide encoding a PB2 protein of a low pathogenic avian influenza virus; a recombinant expression vector including the polynucleotide; an attenuated recombinant virus transformed into the expression vector; and a vaccine composition including the recombinant expression vector. The recombinant expression vector of the present invention enhances chicken embryo productivity of a vaccine strain for preventing influenza, and thereby, having advantages of being capable of being effectively used in enhancing efficacy and productivity of killed vaccines, and additionally, has no pathogenicity for mice and has immunogenicity, and thereby having advantages of being capable of being effectively used as live vaccines. [Reference numerals] (AA) Inserting into the inside of pHW2000-BsmBI

Description

RECOMBINANT EXPRESSION VECTOR FOR PREPARING HIGHLY PRODUCTIVE AND AVIRULENT INFLUENZA VIRUS AND AND USE THEREOF FOR THE PRODUCTION OF INFLUENZA VIRUS

The present invention relates to a PB2 protein and its amino acid sequence that are related to the development of influenza virus, the egg pathogenicity and immunogenicity of the egg, and more particularly, to PB2 protein in a reverse genetics vector system based on the existing PR8 viral genome The present invention relates to a PB2 protein and its amino acid sequence that improves proliferation in a developing egg and eliminates mouse pathogenicity but maintains immunogenicity when a coding polynucleotide is replaced with a PB2 protein coding polynucleotide of the present invention. The inventive egg of the present invention has an advantage of being able to be used for the development of influenza Sadox vaccine and virulence vaccine by enhancing the productivity and safety of the vaccine fetus for prevention of influenza.

Influenza virus belongs to Osomix virus, and has eight single-stranded RNA fragments as negative genomes. From the eight RNA fragments, hemagglutinin (HA), neuraminidase (NA) nucleoside capsid protein (nucleoprotein; NP), matrix protein 1 and 2 (matrix, M _1, M _2), polymerase subunit A, B1 and B2 (polymerase subunit A, B1 &B2; PA, PB1, PB2, respectively), and Nonstructural proteins 1 and 2 (NS1, NS2, respectively) are made.

In the past, the human influenza A vaccine was a unit vaccine in which only HA and NA were purified by inactivating the rearranged strain, which is excellent in proliferation of fetal growth, as formalin, and it is known that the most popular human influenza virus and developmental proliferation A / Puerto Rico / 8/34 (PR8) was mixed and infectious, but recent rearrangements with HA and NA viruses were selected for vaccine production (Kilbourne ED, Future influenza vaccines and the use of genetic recombinants Bull.World Health Organ 1969. 41: 643), recently used reverse genetics technology to adapt the low-temperature PR8 virus or A / WSN / 33 (H1N1) virus, Eight genomes, such as attenuated A / Ann Arbor / 6/60 (H2N2) virus, were cloned into the viral genome transcription vector and cloned into a vector expressing PA, PB1, PB2, Twelve plasmids (Patent No. 0908757) were transfected into a cell line such as 293T to produce the desired virus or transfected eight plasmids in which viral genome transcription occurred in one direction of the vector and mRNA was produced in the other direction (Patent No. 0862758; Vaccine 2002, 20: 3165-3170) to produce a recombinant virus.

Generally, HA and NA genes are amplified by the PCR method from recently introduced viruses, cloned into a reverse genetics vector, and transfected with the remaining 6 genes of PR8 to produce a vaccine strain and inactivate the virus to produce a sadox vaccine. PR8 can not be used as a virulence vaccine due to the presence of pathogenicity in mice. Therefore, the PR8 virus backbone is used but the NS1 gene is deleted and then propagated in vero cells capable of viral propagation without NS1 gene Or [Virology 1998, 252: 324-330; Ann Arbor / 6/60 (H2N2) viruses that have been adapted at low temperatures to reduce pathogenicity (Virology 2003, 306: 18-24; Journal of Virology 2010, 84: 44-51). The nucleotide sequence derived from the HA gene was inserted into the PB1 gene of A / turkey / OH / 313053/04 (H3N2) The HA and NA genes of the virus are co-transfected together to produce attenuated live monocotyledons (Journal of Virology 2011, 85: 456-459).

Although the live monocotyledonous gland can proliferate in vivo to form immunity even when a small number of viruses are inoculated, there is a disadvantage in that the productivity of the vaccine produced in the above manner is significantly lower than that of PR8. Therefore, in the case of the Sadok vaccine, even if the pathogenicity of the mouse remains, the mutation of the internal gene of the PR8 virus or the mutation of the HA and NA amino acids of the vaccine virus may be improved to improve the proliferation in the fetus (Vaccine 2010, 28: 8008-8014 ; Vaccine 2011, 29: 5153-5162; Vaccine 2011, 29: 8032-8041), and virus production technology was reduced to reduce fetal pathogenicity (Archives of Virology 2011, 156: 557-563).

The pathogenicity of the influenza virus and the proliferative properties of the developing egg have been reported in various amino acid mutations in HA, NA, PA, PB1, PB2, NP and NS. Polymerase of influenza virus binds PA at the N-terminal of PB1 with enzyme activity and PB2 at the C-terminal of PB1 to form a complex. It is known that PB1 with enzyme activity has low temperature adaptation and amino acid variations related to the proliferation of avian influenza virus in mammalian cells (Jin et al., 2003, Virology; Journal of General Virology, 2011, Epub ahead .; Virus Research, 2009, 140: 194-198) Mutations associated with proliferation in the fetus are not known.

For the PB2 protein, only the relationship between the host range and the hospital performance is known, but the relationship with the improvement of the proliferative ability of the developing egg is not known. The 622 amino acid of the PB2 protein has been reported to have pathogenicity in humans and mice when the avian influenza virus has glutamic acid but the amino acid changes to lysine (E627K) (Journal of Virology, 1993, 67: 7223-7228 Recently, viruses with high virulence for mice have been reported even with glutamic acid as the 627 amino acid, and E627K and mouse pathogenicity have been reported. (Virology 2011, 410: 1-6; Virus Research, 2009, 44: 123-43). In addition, the amino acid sequences of the 627 amino acids as well as other parts of the amino acid sequence of the 627- Virology, 2010, 401: 1-5).

The PA protein is important for the interaction of PB1 and PB2 and is known to be related to the pathogenicity of the avian influenza virus (mammalian), the pathogenicity to ducks and the fetal pathogenicity (J of Virology, 2011, 85: 7020-7028; Virus Research , 2011, 155: 325-333; Infection, Genetics and Evolution, 2011, 11: 1790-1797; Ilyushina et al., 2010, J of Virology, 84: 8607-8616; Journal of Virology, 2011, 85: 2180-2188 ) Mutations related to proliferation in the fetus are not known.

On the other hand, 01310 is a virus registered in the domestic patent (patent No. 0708593) because it has high productivity and low pathogenic H9N2 subtype avian influenza virus and it is related to mutation in HA and NA protein (Journal of Veterinary Science 2008, 9: 67-74), but the relevance of PB2, PB1, and PA proteins is unknown.

Therefore, the inventors of the present invention found that a vector system based on the H1N1 avian influenza virus (for example, PR8 virus) system for proliferation in the developmental field superior to the PR8 virus and having no pathogenicity in a mouse (mammalian) , Recombinant viruses were prepared by substituting the coding polynucleotide of PB2, PB1, and PA protein of PR8 with one of the coding polynucleotides of PB2, PB1, and PA proteins derived from the pathogenic avian influenza virus, respectively. Recombinant viruses were prepared from these viruses The recombinant virus substituted with PB2 protein coding polynucleotide is excellent in proliferation in developmental cells, safe from lack of mouse pathogenicity, but possesses immunogenicity and is useful as an effective vaccine, thereby completing the present invention.

Accordingly, one example of the present invention provides a use for use in the development of the developing egg of the H1N1 strain of avian influenza virus (for example, PR8 virus) of the PB2 protein coding polynucleotide derived from the low-pathogenic avian influenza virus and for the removal of the mouse (mammalian pathogenic) do.

Another example provides a recombinant expression vector comprising a H1N1 strain of avian influenza virus (e. G., PR8 virus) genome in which the PB2 protein coding polynucleotide has been replaced with a PB2 protein coding polynucleotide derived from a low pathogenic avian influenza virus.

Another example provides a recombinant H1N1 strain of avian influenza virus (e. G., The PR8 virus) in which a PB2 protein coding polynucleotide has been replaced with a PB2 protein coding polynucleotide derived from a low pathogenic avian influenza virus.

Another example provides a vaccine composition comprising the recombinant H1N1 strain of avian influenza virus (e.g., PR8 virus) as an active ingredient.

The A / Puerto Rico / 8/34 (H1N1) virus (hereinafter referred to as 'PR8 virus', NC_002016 to NC_002023 gene) used in the present specification is a virus widely used for the vaccine production of H1N1 strain avian influenza virus, Which has a problem of poor safety, but is used in spite of potential risks due to its good proliferation.

Thus, the present inventors have completed the present invention by further improving the proliferation of the PR8-based vector system in the fetus and maintaining immunogenicity while eliminating potential pathogenicity.

The present invention relates to a method for screening a PB2 protein coding polynucleotide from a recombinant strain in which PB2, PB1, or PA protein coding polynucleotide derived from PR8 virus is replaced with PB2, PB1, or PA protein coding polynucleotide derived from a pathogenic avian influenza virus, respectively, A recombinant strain substituted with a virus-derived vector provides a composition of a vector system for producing an influenza vaccine excellent in proliferating cell proliferation, and having no maternal and mouse pathogenicity but immunogenicity. That is, the present invention suggests that the PB2 protein coding gene in the H1N1 strain of avian influenza virus is related to the high productivity of the developing eggs of influenza virus.

As described above, the composition of the reverse genetics vector system for producing a highly proliferating influenza virus in the developmental field of the present invention is not limited to the PR8-based reverse genetics vector system but may be applied to all influenza reverse genetics vector systems Can be applied.

First, one example of the present invention provides for the development and / or attenuation of the developing egg of the H1N1 strain avian influenza virus of the PB2 protein coding polynucleotide of the pathogenic avian influenza virus. Accordingly, there is provided a composition for the production of attenuated H1N1 strain avian influenza virus, specifically attenuated developmental oocyte hyperproliferative PR8 virus, comprising an attenuated developmental egg containing PB2 protein coding polynucleotide of the pathogenic avian influenza virus as an active ingredient. In another aspect, an attenuated H1N1 strain of avian influenza virus, specifically attenuated, comprising a step of replacing the PB2 gene of the H1N1 strain of avian influenza virus with a PB2 protein coding polynucleotide of an avirulent avian influenza virus A method for the production of hypertrophic PR8 virus is provided.

A / chicken / Korea / KBNP-0028/2000 (H9N2) (registered patent No. 0708593, hereinafter referred to as KBNP-0028), which is a low-pathogenic avian influenza virus, 2001 (H9N2) (National Veterinary Research and Quarantine Service, Department of Algae Diseases; Registered Patent No. 0790801, hereinafter referred to as "01310"), and more specifically 01310 strain. The 01310 strain is a domestic isolate isolated as in Example 1 of Patent Registration No. 0790801 and is currently in a repository in the Department of Algae and Disease Control at the Agriculture, Forestry and Fisheries Inspection Headquarters (GenBank accession No. JX094853-JX094856, JX094858-JX094861). The 01310 strain is classified as a pathogenic virus and is unable to deposit in the depository, and is therefore kept in the avian disease section.

The PB2 protein of the pathogenic avian influenza virus, e.g., strain 01310, may comprise one or more of the following features:

Wherein the amino acid 66 is isoleucine (I),

The amino acid 88 is lysine (K),

When the amino acid 109 is isoleucine (I),

When the amino acid 133 is isoleucine (I),

When the amino acid 157 is arginine (R),

Amino acid 286 is glycine (G),

When the amino acid 315 is isoleucine (I),

373 amino acid is leucine (L)

The amino acid at position 451 is isoleucine (I),

The amino acid 575 is valine (V), and

If amino acid 674 is alanine (A)

When the H1N1 strain of avian influenza virus, specifically the PR8 virus, is a PB2 protein-encoding polynucleotide and has the PB2 protein coding polynucleotide encoding the PB2 protein of the low-pathogenic avian influenza virus having the above-mentioned characteristics, Disappear.

For example, the PB2 gene of strain 01310 has the nucleotide sequence of SEQ ID NO: 1, from which a PB2 protein having the amino acid sequence of SEQ ID NO: 2 is produced.

More specifically, in the present invention, the PB2 protein coding polynucleotide of the H1N1 strain of avian influenza virus, specifically, the minor pathogenic avian influenza which is substituted with the PB2 gene of the PR8 virus, may be one encoding a PB2 protein having the amino acid sequence of SEQ ID NO: 2 .

In an embodiment, the PB2 protein coding polynucleotide of the pathogenic avian influenza virus may be a coding polynucleotide of PB2 protein (SEQ ID NO: 2) derived from strain 01310 having the nucleotide sequence of SEQ ID NO: 1 as the PB2 gene.

In another example, the invention comprises a PB2 protein coding polynucleotide in a H1N1 strain of avian influenza virus, specifically a genome of the PR8 virus (including NC_002016 to NC_002023 genes), substituted with a PB2 protein coding polynucleotide of an avirulent avian influenza virus To provide a recombinant expression vector. The recombinant expression vector is useful as a vector system for virus production with high fetal growth and low virulence.

More specifically, the recombinant expression vector may be a polymerase B1 (PB1) -coding polynucleotide of the H1N1 strain of avian influenza virus, specifically of the PR8 virus (including the gene of NC_002016 to NC_002023), a polymerase A (PA) -coding polynucleotide (NA) coding polynucleotide, and a matrix protein (M) -coding polynucleotide, an unconjugated protein (NS) -coding polynucleotide, a nucleic acid encoding a polynucleotide , And the PB2 protein coding polynucleotide of the pathogenic avian influenza virus.

The PB2 protein encoded by the PB2 protein coding polynucleotide of the pathogenic avian influenza virus may comprise one or more of the following features:

Wherein the amino acid 66 is isoleucine (I),

The amino acid 88 is lysine (K),

When the amino acid 109 is isoleucine (I),

When the amino acid 133 is isoleucine (I),

When the amino acid 157 is arginine (R),

Amino acid 286 is glycine (G),

When the amino acid 315 is isoleucine (I),

373 amino acid is leucine (L)

The amino acid at position 451 is isoleucine (I),

The amino acid 575 is valine (V), and

If amino acid 674 is alanine (A)

As described above, the PB2 protein coding polynucleotide of the pathogenic avian influenza virus may be a PB2 protein coding polynucleotide derived from the strain 01310, and more specifically, a PB2 protein coding polynucleotide having the amino acid sequence of the PB2 protein having the amino acid sequence of SEQ ID NO: A coding polynucleotide of the protein or a polynucleotide having the nucleotide sequence of SEQ ID NO: 1.

For example, the recombinant expression vector of the present invention may be represented by the cleavage map of FIG. 3, but is not limited thereto.

Another example of the present invention is a H1N1 avian influenza virus mutant strain in which a PB2 protein coding polynucleotide in the genome of the H1N1 strain of avian influenza virus is substituted with a PB2 protein coding polynucleotide of an avirulent avian influenza virus, Wherein the PB2 protein coding polynucleotide is replaced with a PB2 protein coding polynucleotide of a pathogenic avian influenza virus.

The recombinant virus may be a recombinant H1N1 strain of avian influenza virus transformed with the recombinant expression vector, specifically recombinant PR8 virus transformed with the recombinant expression vector. For example, the recombinant PR8 virus can be obtained by substituting the PB2 protein coding polynucleotide in the PR8 genome with the coding polynucleotide of the PB2 protein (SEQ ID NO: 2) of strain 01310 or the PB2 gene of SEQ ID NO: 1, more specifically the PB2 protein 2) with a coding polynucleotide (rPR8-01310 (PB2)) (KCTC 12076BP).

The recombinant virus is very useful for the production of an influenza virus vaccine which is excellent in propagation (embryonic development) and is safe and effective because it has excellent immunity and low or no pathogenicity in birds such as chickens and ducks as well as mammals such as mice. Especially, it can be used as a virulent vaccine as well as a sadox vaccine due to its superiority and excellent safety.

Accordingly, another example of the present invention provides a bird flu virus vaccine comprising the above-described developmental (maternal fetus) high productivity and recombinant virus of algae and mammalian pathogen as an active ingredient. The recombinant virus is particularly a strain (PB2) in which the PB2 protein coding polynucleotide in the PR8 virus (NC002016 to NC002023) genome is replaced with the coding polynucleotide of the coding polynucleotide of PB2 protein (SEQ ID NO: 2) of strain 01310 ) ≪ / RTI > (KCTC 12076BP).

The vaccine is not only excellent in proliferation but also immunogenicity with low or no pathogenicity to mammals and birds including mice, so that it can be used as a poison vaccine as well as a sadox vaccine.

The vaccine is applicable not only to birds such as chickens and ducks but also mammals including humans, pigs, dogs, mice, and the like.

In addition, the present invention provides a composition for diagnosing avian influenza virus, which comprises high productivity of the developing bovine (fetus) and antisera against avian and mammalian pathogenic recombinant viruses, and a diagnostic kit. The recombinant virus may be rPR8-01310 (PB2) (KCTC 12076BP). The antiserum may be obtained from algae or mammals, and the subject to be diagnosed may be algae or mammals.

Hereinafter, the present invention will be described in more detail.

Infectious viral infections in Korea were infected with Korean pathogen-free avian influenza virus 01310 and KBNP-0028 (KCTC 10866BP, patent registration No. 0708593) in 10 to 11 days specific pathogen free (SPF) After amplification of PB2, PB1, PA protein coding polynucleotide of 01310 strain and PB2 of KBNP-0028 (KCTC 10866BP) by RT-PCR, the amplified product was cut out by electrophoresis on agarose gel and purified. Respectively. PB1, and PA protein coding polynucleotides of the 01310 strain in place of the PB2, PB1, and PA protein coding polynucleotides of the PR8 virus-based reverse genetics vector system, and KBNP- The recombinant viruses rPR8-01310 (PB2), rPR8-01310 (PB1), rPR8-01310 (PA) and rPR8-KBNP-0028 (PB2) were prepared by reverse genetics by substituting one PB2 of each of the recombinant viruses (KCTC 10866BP) . The proliferative activity of recombinant viruses (50% embryo infection dose, EID ₅₀ / ml) in the fetus was 10 ^8.8 , rPR8-01310 (PB1) was 10 ^7.6 , and rPR8-01310 (PA) was 10 ^8.5 and rPR8-KBNP-0028 (PB2) showed 10 ^8.5 EID ₅₀ / ml, respectively. However, rPR8-01310 (PB2) showed 10 ^9.4 EID ₅₀ / ml compared to rPR8 and showed a higher proliferative activity than rPR8 (Table 1).

The reason why rPR8-01310 (PB2) with PB2 of strain 01310 of the present invention showed higher proliferative egg proliferation than rPR8 and rPR8-KBNP-0028 (PB2) viruses with PB2 protein coding polynucleotide of PR8 and KBNP-0028 As a result of comparison of PB2 amino acids of 01310, PR8 and KBNP-0028, 01310 showed 66 amino acids instead of methionine instead of isoleucine, 88 amino acid, lysine instead of arginine and valine as amino acid 109 Isoleucine, isoleucine in place of valine in position 133, arginine in lieu of lysine in position 157, glycine in place of serine in position 286, isoleucine in place of methionine in position 315, isoleucine in place of isoleucine in position 373, , Amino acid 451 isoleucine instead of valine, amino acid 575 is valine instead of methionine, amino acid 674 is Tranino or glycine instead of Allah With it were different (Table 2).

To investigate the mouse pathogenicity of the rPR8, rPR8-01310 (PB2), rPR8-01310 (PB1) and rPR8-KBNP-0028 (PB2) viruses of the present invention, 4-5 week old BALB / c mouse female 5 The rPR8-01310 (PB2) and rPR8-KBNP-0028 (PB2) were inoculated into the nasal cavity with 10 ⁶ EID ₅₀ / mouse inoculated with water, (PB1) recombinant virus showed a significant decrease in body weight with the dead rats, indicating that the rPR8 and rPR8-01310 (PB1) recombinant viruses showed pathogenicity to mice (Fig. 1) . In order to examine the immunogenicity of rPR8-01310 (PB2) of the present invention, mice vaccinated with the virus and mice not vaccinated with the virus were bred for 2 weeks and then rPR8 virus was infected with rPR8- The mice vaccinated with 01310 (PB2) and rPR8-KBNP-0028 (PB2) did not show any reduction in body weight or mortality, but vaccine efficacy was observed. In the control group, severe weight loss was observed together with mortality, (Fig. 2).

RPR8, rPR8-KBNP-0028 ( PB2) a 10 ^-3 dilution in I powerful route to 10-11 day-old SPF balyukran in order to investigate the pathogenicity of the fetus based on the recombinant virus of the present invention, rPR8-01310 (PB2) virus (Table 3). There was no pathogenicity to the embryos, as there were no lesions or deaths such as hemorrhage or hemorrhage.

The present invention provides a recombinant PR8 virus having no or low virulence and excellent immunogenicity in order to solve the problem of pathogenicity in the production of a vaccine using the PR8 virus. Fetal) high productivity, mammalian high immunogenicity, and algae and mammalian pathogenicity, which are useful as vaccines as well as poisoned sods.

FIG. 1A is a graph showing the results of immunohistochemical staining for A / chicken / Korea / KBNP-0028/2000 (H9N2) (Patent No. 0708593, hereinafter referred to as KBNP-0028), A / chicken / Korea / 01310/2001 (H9N2) (PB2), rPR8-01310 (PB2), rPR8-01310 (PB1), recombinant viruses prepared by replacing PR8 PB2 and PB1 with the PB1 gene, BALB / c mouse mortality compared to the parental virus, rPR8.
Figure 1b shows the effect of the same virus on the BALB / c mouse body weight compared to the parent virus rPR8.
FIG. 2 shows the results of comparing the defensive ability by inoculating rPR8-01310 (PB2), rPR8-KBNP-0028 (PB2) virus into BALB / c mice in a PR8 vector system and inoculating rVR8, a homologous virus, two weeks later.
Fig. 3 is an illustration of cleavage map of an expression vector for transformation of PR8 virus.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: Low pathogenic  Origin of avian influenza virus PB2 , PB1 , PA  Protein coding The polynucleotide  Recombination PR8  virus Operation

1-1: PR8  Preparation of the virus

The PR8 virus used as the parent virus of the recombinant virus in the present invention was prepared in the following manner.

Specifically, pHW191-PB2, pHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW196-NA, pHW197-M corresponding to the whole cDNA of PR8 (A / Puerto Rico / 8 / , And plasmid for pHW198-NS reverse genetics (Vaccine 2002, 20: 3165-3170). And Dr. Webster from Jude Children's Research Hospital. 293T cells (Life Resource Center, KCTC) were inoculated into 6-well cell culture dishes in MEM (GIBCO BRL) medium containing 5% (v / v) FBS at 2 × 10 ⁶ cells / And then added to each well, followed by adhering for 3 to 4 hours. After the medium was removed, 2 ml of Opti-MEM medium (Invitrogen Co. USA) was added.

Each of the 8 plasmids prepared above was put into a 1.5 ml tube in an amount of 300 ng each, Add Opti-MEM medium to final 25 μl, add 6 μl of PLUS reagent (Invitrogen Co. USA) and 69 μl Opti-MEM medium to another 1.5 ml tube, mix in a 1.5 ml tube containing the plasmid And the mixture was reacted at room temperature for 15 minutes. During the waiting period, 96 μl of lipofectamine (Invitrogen Co) and Opti-MEM were mixed and reacted for 15 minutes. 100 μl of the mixture was added to the plasmid-containing tube, followed by further reaction for 15 minutes.

The resultant reaction product was added to each well containing the 293T cells in an amount of 100 占 퐇. After culturing the 6-well culture container with 5% CO ₂ at 37 ° C for 20 hours, 10 μg (2.5 μg / 4 μl) of trypsin per well was added, and the supernatant was harvested after 24 hours. (Sunrise Co., NY) was inoculated with 200 ul of the above-harvested stock solution into the intestinal tract. When the inoculated eggs were cultured at 37 ° C for 3 days, the intercellular fluid was harvested and the hemocyte aggregation titers were measured when the hemocyte aggregates were obtained. RNA was extracted from the viruses grown in the eggs by the same method and diluted 100 times, After confirming that the PR8 virus (including NC_002016 to NC_002023) was properly constructed, it was stored at -70 ° C. The thus prepared strain was used as PR8 virus strain in the following experiment. rPR8 means a recombinant virus and has the same gene composition as wild type PR8 and can be mixed with 'PR8'.

1-2: Virus growth

Each sample (PR8 (Example 1-1), KBNP-0028 (A / chicken / Korea / KBNP-0028/2000 (H9N2), KCTC 10866BP, patent registration No. 0708593), 01310 (Total of 27 bovine sponges) were inoculated into three allantoic cavities of SPF developmental field (Sunrise Co., NY), and the cells were incubated at 37 ° C After culturing for 3 days, the allantoic fluid was harvested and the following plate hemagglutination test was performed. The 20 쨉 l of the umbilical fluid and the SPF developmental cells (Sunrise Co., NY) inoculated with each of the above samples were hatched Twenty microliters of 0.1% (w / v) chicken erythrocytes from chicken chickens were spotted on a glass plate and mixed with plate hemagglutination test. Positive samples were stored at -70 ℃.

1-3: Reverse transcriptase - Polymerase chain reaction , gene Cloning , Sequencing and analysis

For RNA isolation, RNA was isolated from 150 발 of membrane growth culture of rPR8, 01310, KBNP-0028 virus using Viral Gene spin kit (iNtRON Co. Ltd., Seongnam, Korea) according to the method provided by the manufacturer After that, a one-step RT-PCR kit of QIAGEN and a primer for influenza virus genome amplification (PB2 amplification primer Ba-PB2-1, Ba-PB2-2341R; PB1 amplification primer, BmPB1-1, Bm-PB1-2341R; Eight genomes of the virus were amplified using primers PA amplification, Bm-PA-1, Bm-PA-2233R, Hoffmann et al., Archives Virol, 2001). 4 μl of 5X one step RT buffer, 4 μl of dNTP (2.5 mM), 1 μl of forward and reverse primers (10 pmol / μl) for each gene amplification, 4 μl of one-step enzyme mix, 4 μl of RNA, After reacting at 50 ° C for 30 minutes with a PCR device (C-1000, Bio Rad Laboratories Inc., USA), the reaction mixture was heated at 95 ° C for 15 minutes and then reacted at 94 ° C for 20 seconds, The reaction was repeated 30 times for 30 seconds at -72 ° C for 7 minutes, followed by reaction at 72 ° C for 7 minutes. The obtained amplification product was electrophoresed on an agarose gel and purified using a gel extraction kit (QIAGEN Co., USA), and the base sequence was determined using an ABI3777 automatic base sequence analyzer (Macrogen, Seoul, Korea). SEQ ID NO: 1 is a polynucleotide encoding a PB2 protein of strain 01310, SEQ ID NO: 2 is an amino acid sequence of a PB2 protein, SEQ ID NO: 3 is a PB1 protein coding polynucleotide, SEQ ID NO: 4 is an amino acid sequence of a PB1 protein, SEQ ID NO: 5 is the 01310 PA protein coding polynucleotide, SEQ ID NO: 6 is the amino acid sequence of the PA protein, SEQ ID NO: 7 is the PB2 protein coding polynucleotide of KBNP-0028 and SEQ ID NO: 8 is the amino acid sequence of the PB2 protein .

1-3. Production of recombinant virus

Hoffmann's reverse genetics vector system (Patent No. 0862758) was used to generate recombinant influenza virus.

Specifically, PB2, PB1, PA of 01310 strain amplified in Example 1-2 and PB2 protein coding polynucleotide of KBNP-0028 were cloned into a TOPO-TA cloning vector (Invitrogen Co. USA) to obtain M13F / M13R primer , 5'-GTAAAACGACGGCCAG-3 '(SEQ ID NO: 9), M13R, 5'-CAGGAAACAGCTATGAC-3' (SEQ ID NO: 10), 20U / 2μl of BsmBI (NEB Inc, USA), NEBuffer 10 μg / 3 μl of a pHW2000 plasmid (Korean Patent No. 0862758, obtained from the National Veterinary Research and Quarantine Service, Dr. Webster, Dr. Webster, with the consent of Dr. Hoffman), sterilized tertiary 13 μl of distilled water was mixed and incubated at 50 ° C for 1.5 hours. The DNA was electrophoresed on an agarose gel, and the DNA was purified with a gel extraction kit (QIAGEN Co.).

top10 competent cell, Invitrogen Co. USA)에 transformation 한 후 염기서열을 분석하여 유전자의 염기서열의 돌연변이가 없는 정확한 클론을 선발하여 플라스미드를 추출하였다. Also in the same way as the processing BsmBI to rise agarose gel electrophoresis the vector pHW2000 6㎕ with T4 DNA referred to in this NS 2㎕ purified as described above in respect to the plasmid pHW2000 (1,000,000 U / ㎕; NEB Inc , USA) 1 Mu] l of 10X reaction buffer (NEB Inc, USA) was added and allowed to stand at room temperature for 2 hours. Escherichia coli coli , OneShot

top10 competent cell, Invitrogen Co. USA), and then the nucleotide sequence was analyzed to select an exact clone without mutation of the nucleotide sequence of the gene to extract the plasmid.

In Example 1-1, PHW192-PB1, pHW193-PA, pHW194-HA, pHW195-NP, pHW196-NA, pHW197-M and pHW198-NS plasmids for reverse genetics (Vaccine 2002, PBW, pHW192-PB1, or pHW193-PA in a plasmid in which 8 genes of PR8 virus were inserted into pHW2000 (see Patent Document 10-0862758) 293T cells (Life Resource Center, KCTC) were inoculated into 6-well cell culture vessels in MEM containing 5% (v / v) FBS in order to produce recombinant viruses with PB2 plasmids of PB2, PB1, PA or KBNP- (GIBCO BRL) medium in an amount of 2 × ¹⁰ 6/2 ml, added to each well, and allowed to adhere for 3-4 hours. After the medium was removed, 2 ml of Opti-MEM medium (Invitrogen Co. USA) was added.

One of the prepared plasmids (7 plasmids corresponding to the 7 genes except the PB2, PB1, or PA gene of the PR8 virus plus one corresponding to the PB2, PB1, or PA gene of the homologous (PR8) or heterologous virus (01310 and KBNP- 0028) Plasmid) were put into a 1.5 ml tube so that the amount of each plasmid was 300 ng, Add Opti-MEM medium to final 25 μl, add 6 μl of PLUS reagent (Invitrogen Co. USA) and 69 μl Opti-MEM medium to another 1.5 ml tube, mix in a 1.5 ml tube containing the plasmid And the mixture was reacted at room temperature for 15 minutes.

During the waiting period, 96 μl of lipofectamine (Invitrogen Co) and Opti-MEM were mixed and reacted for 15 minutes. 100 μl of the mixture was added to the plasmid-containing tube, followed by further reaction for 15 minutes.

The resultant reaction product was added to each well containing the 293T cells in an amount of 100 占 퐇. After culturing the 6-well culture container with 5% CO ₂ at 37 ° C for 20 hours, 10 μg (2.5 μg / 4 μl) of trypsin per well was added, and the supernatant was harvested after 24 hours. (Sunrise Co., NY) was inoculated with 200 ul of the above-harvested stock solution into the intestinal tract. The inoculated cells were incubated at 37 ° C for 3 days and then the umbilical fluid was harvested. When the cells were aggregated, the hemagglutination titers were measured. The cells were diluted 100 times and RNA was extracted from the viruses grown in the embryonated eggs. PB1 and PA genes were determined to confirm that the virus was properly constructed and stored at -70 ° C.

As a result of this example, rPR8-01310 (PB2) (PB2 protein coding polynucleotide of PR8 virus was replaced with PB2 protein coding polynucleotide (SEQ ID NO: 1) of 01310 strain), rPR8-01310 (SEQ ID NO: 3) of the 01310 strain), rPR8-01310 (PA) (the PA protein coding polynucleotide of the PR8 virus is the PA protein coding polynucleotide of the 01310 strain (SEQ ID NO: 5)), the coding polynucleotide is replaced with the PB1 protein coding polynucleotide ), RPR8-KBNP-0028 (PB2) (PB2 protein coding polynucleotide of PR8 virus was replaced with PB2 protein coding polynucleotide of KBNP-0028 (SEQ ID NO: 7)) and rPR8 rPR8-01310 PB2: SEQ ID NO: 1) was deposited on Nov. 18, 2011 with the deposit number KCTC 12076BP at the Life Resource Center in Daejeon.

1-4. virus Potency  Measure

To measure the proliferative activity (50% embryo infection dose, EID 50 / ml) in the recombinant virus produced by the fetus based on the above, decimal dilution to each of the recombinant virus in phosphate buffer 10 ^-1 to 10 ^-9 , And 100 μl of the urethral catheter pathway was inoculated into five SPF developmental bouts of 10-11 days of age at each dilution. After incubation for 3 days, the urothelial fluid was harvested, and the cell viability was confirmed by red blood cells of the chicken, and the virus titer (EID ₅₀ / ml) was measured according to the Reed-Muench equation.

The resulting virus-derived fetal infectious titer (EID ₅₀ / ml (log 10)) is shown in Table 1 below. As can be seen in Table 1, the fetal infectious component of rPR8 showed 10 ^8.8 EID ₅₀ / ml, and both rPR8-01310 (PA) and rPR8-KBNP-0028 (PB2) showed 10 ^8.5 EID ₅₀ / ml Similar to rPR8, rPR8-01310 (PB1) was 10 ^7.6 EID ₅₀ / ml, indicating a decrease in fetal growth. However, rPR8-01310 (PB2) showed 10 ^9.4 EID ₅₀ / ml, indicating that the proliferative activity of the fetus was significantly increased compared to other recombinant viruses including rPR8.

The amino acid sequence of 01310 PB2 protein amino acid sequence and the amino acid sequence of rPR8 and KBNP-0028 PB2 proteins were compared with those of 01310 PB2 in order to investigate the amino acid sequence related to the improvement of fetal growth. 01310 PB2 of the 01310 strain was identified as PR8, KBNP- In contrast, the amino acid 66 is isoorucine instead of methionine, lysine instead of arginine as amino acid 88, isoleucine instead of valine as amino acid 109, isoleucine instead of valine as amino acid 133, arginine instead of lysine as amino acid 157, amino acid 286 Glycine instead of serine, isoleucine instead of methionine as amino acid 315, lysine as lysine instead of isoleucine as 373 amino acid, isoleucine as valine as 451 amino acid, valine as methionine instead of methionine as 575 amino acid, threonine as amino acid 674 They had alanine instead of glycine and showed a difference.

Example  2: Low pathogenic  Origin of avian influenza virus PB2 , PB1 , PA  Protein coding The polynucleotide  Recombination PR8  Viral pathogenicity and immunogenicity measurement

2-1. BALB / c Pathogenicity measures for mice

BALB / C mice (6 weeks old) were anesthetized with 15 mg / kg of zoletil (Virbac SA, France) by rinsing with rPR8, rPR8-01310 (PB1) and rPR8-01310 Female, Coatec Co., Ltd.) were inoculated with 10 ⁶ EID ₅₀ into 5 nasal passages, respectively. After that, the presence or absence of dead skin was examined every day, and the body weight was measured and shown in Figs. 1A and 1B, respectively. As a result, the mortality rate of rPR8 and rPR8-01310 (PB1) was 100%. In these recombinant strains, a significant decrease in body weight was observed, but in rPR8-01310 (PB2) and control (inoculation with phosphate buffer instead of virus) , And no decrease in body weight was observed, indicating no pathogenicity (see Figs. 1A and 1B).

2-2. BALB / c Measuring immunogenicity for mouse

The recombinant virus rPR8-01310 (PB2) and rPR8-KBNP-0028 (PB2) to 1X10 ⁶ were inoculated intranasally attack a BALB / C mouse was observed for 2 weeks (5 week-old female, Tec Corporation cores) rPR8 virus 1X10 ⁶ dogs . Mice inoculated with rPR8-01310 (PB2) and rPR8-KBNP-0028 (PB2) were healthy without changes in body weight before and after the rPR8 attack. However, rPR8-01310 (PB2) and rPR8-01310 confirming the existence of immunogenicity of rPR8-KBNP-0028 (PB2), it was judged to be useful for the development of a virulence vaccine as well as a Sodok vaccine (see Fig. 2).

2-3. Chick  Pathogenic measurement

The recombinant virus was diluted 10 ^-3 with a phosphate buffer solution and inoculated into the umbilical cord at an age of 10 to 11 days in a SPF broth (Sunrise Co., NY) in an amount of 200 쨉 l, cultured at 37 째 C for 3 days, And 3 days after storage at 4 ° C for 12-24 hours. The lesions were observed in the fetuses and the lesions such as hemorrhage or redness. The results obtained are shown in Table 3. As can be seen in Table 3, rPR8-01310 (PB2), like the other two viruses, did not cause any maternal mortality for 3 days and could be used as a vaccine to meet OIE recommendations

Since the maternal fetus, which is dead in the midst of production of vaccine using the fetus, can not be used for vaccine production, the virulence of the fetus is associated with low virulence for vaccine birds as well as vaccine productivity. Therefore, the 01310 PB2 protein coding polynucleotide, It can be useful for making various vaccine stocks with high productivity and safety.

virus Number of development Number of deaths after our inoculation 1 day 2 days 3 days rPR8 5 0 0 0 rPR8-01310 (PB2) 5 0 0 0 rPR8-KBNP-0028 (PB2) 5 0 0 0

Korea Biotechnology Research Institute KCTC12076BP 20111118

<110> SNU R & DB FOUNDATION          REPUBLIC OF KOREA (Management: Ministry for Food, Agriculture, Forestry and Fisheries. National Veterinary Research and Quarantine Service (NVRQS)) <120> RECOMBINANT EXPRESSION VECTOR FOR PREPARING HIGHLY PRODUCTIVE AND          AVIRULENT INFLUENZA VIRUS, AND USE THEREOF <130> DPP20116487KR <160> 10 <170> Kopatentin 1.71 <210> 1 <211> 2280 <212> DNA <213> Artificial Sequence <220> <223> PB2 gene of 01310 <400> 1 cgagatactg acaaaaacca ctgtggacca tatggccata atcaagaaat acacatcggg aagacaggag 120 aagaaccccg ctctcagaat gaaatggatg atggcaatga aatacccgat tacagctgac 180 aaaagaataa tggagataat ccctgaaaga aatgagcaag gtcaaactct ctggagcaaa 240 acgaatgatg ctggatcaga taaggtaatg gtatcacctc tggctgtgac gtggtggaat 300 agaaatggac caacaacaag cacaatccat tatcccaagg tatataaaac ttactttgag 360 aaggttgaaa ggttgaaaca cggaaccttt ggccccattc atttccgaaa tcaagtcaaa 420 atacgccgca gggttgacat aaacccgggc catgcagacc tcagtgctag agaagcacaa 480 gatgttatca tggaggtcgt tttcccgaat gaggttggag ccaggatact gacatcagaa 540 tcacaattga caataacaaa ggaaaagaaa gaagaactcc aggattgtaa gattgctcct 600 ttaatggtgg catacatgtt agaaagagaa ctggttcgca agaccagatt cctaccagtg 660 gctggcggga caagcagcgt gtatatagaa gtactacatt tgactcaagg aacctgctgg 720 gagcagatgt acacaccagg aggggaagta aggaatgatg atgttgacca gagtttgatc 780 attgctgcta gaaacattgt aaggagagca acagtatcag cagacccatt ggcctcgctc 840 ttggagatgt gtcatggtac acaaataggc ggaataagaa tggtagacat ccttagacaa 900 aacccaacag aagagcaagc cgtggatata tgcaaggcag caataggcct aagaatcagt 960 tcatctttca gctttggagg tttcactttc aaaagaacaa gtgggtcttc tgtcaagaag 1020 gaagaagaag tgcttacagg caacctccag acattgaaaa taagagtgca tgagggatat 1080 gaggaattca caatggttgg gcgaagagca acagccctcc taaggaaagc aaccagaagg 1140 ctgattcaac tgatagtaag tgggagagac gaacaatcaa tcgctgaagc gatcattgta 1200 gcaatggtat tttcacaaga ggactgcatg ataaaggcag tccgaggtga tttgaacttc 1260 gtgaacagag cgaaccagcg actgaacccc atgcaccaac ttctgaggca cttccaaaaa 1320 gatgcaaaag tgttgtttca gaactgggga attgaaccca tcgacaatgt catggggatg 1380 attggaatat tgcccgacat gactcccagc acagagatgt cactaagagg agtgagagtc 1440 agcaaaatgg gagtggacga atactctagc actgaaagag tggttgtgag tattgatcgt 1500 ttcttaaggg ttcgagatca gagggggaac atacttctat cccctgaaga agttagtgag 1560 acacaaggga cggaaaagtt aacaataaca tattcatcgt ctatgatgtg ggagattaac 1620 ggcccggaat cagtgctagt taacacatac caatggatca ttaggaattg ggagactgtg 1680 aagattcagt ggtctcaaga tcctactatg ttatacaata aggtggaatt tgaacccttt 1740 caatctctgg tacctaaagc tgccagaggc caatatagtg gatttgtgag aacgctattc 1800 caacaaatgc gtgatgtact gggaacattt gacactgttc agataataaa gctgctacca 1860 tttgccgcag ccccaccaga gcagagtagg atgcagtttt cttctctgac tgtgaatgta 1920 agaggctcag gaatgagaat ccttgtgaga ggcaactccc ctgtgtttaa ttacaacaag 1980 gcaaccaaga ggcttacagt cctcgggaag gatgcaggtg cactcacaga agacccagat 2040 gaaggaacag caggagtgga atccgcagta ttgagaggat tcctaattct aggcaaagaa 2100 gacaagagat acggaccagc attgagcatc aacgaattga gcaatcttgc gaaaggggag 2160 aaagctaatg tattgatagg gcaaggagac gtagtgttgg taatgaaacg gaaacgggac 2220 tctagcatac ttactgacag ccagacagcg accaaaagaa ttcgaatggc catcaattag 2280                                                                         2280 <210> 2 <211> 759 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PB2 protein of 01310 <400> 2 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr   1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Lys              20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys          35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Lys Arg Ile Met      50 55 60 Glu Ile Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys  65 70 75 80 Thr Asn Asp Ala Gly Ser Asp Lys Val Met Val Ser Pro Leu Ala Val                  85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Ile His Tyr Pro             100 105 110 Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly         115 120 125 Thr Phe Gly Pro Ile His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg     130 135 140 Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Arg Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile                 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Glu             180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu         195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr     210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp                 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val             260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Gly Thr Gln         275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu     290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Ile Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser                 325 330 335 Ser Val Lys Lys Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu             340 345 350 Lys Ile Arg Val His Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg         355 360 365 Arg Ala Thr Ala Leu Leu Arg Lys Ala Thr Arg Arg Leu Ile Gln Leu     370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly                 405 410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His             420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn         435 440 445 Trp Gly Ile Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu     450 455 460 Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val                 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu             500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr         515 520 525 Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser     530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Val Glu                 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ala Arg Gly Gln Tyr             580 585 590 Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly         595 600 605 Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala     610 615 620 Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe                 645 650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala             660 665 670 Gly Ala Leu Thr Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser         675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr     690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu 705 710 715 720 Lys Ala Asn Val Leu Ile Gly Gln Gly Asp Val Val Leu Val Met Lys                 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys             740 745 750 Arg Ile Arg Met Ala Ile Asn         755 <210> 3 <211> 2274 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > PB1 gene of 01310 <400> 3 atggatgtca atccgacttt acttttcttg aaagtgccag cgcaaaatgc tataagtacc 60 acattccctt atactggaga ccctccatac agccatggaa caggaacagg atacaccatg 120 gacacagtca acagaacaca tcaatactca gaaaagggga aatggacaac aaacaccgaa 180 actggagcac cccaactcaa tccgattgat ggaccactac ctgaagataa cgagccgagt 240 gggtatgcac aaacagattg tgtattggag gcaatggctt tccttgaaga atcccaccca 300 gggatcttcg aaaattcgtg tcttgaaacg atggaagttg ttcagcaaac aagagttgat 360 aaattgactc aaggtcgcca gacctatgac tggacattga atcggaacca gccagctgca 420 actgctttaa ccaacactat agaggtcttc agatcgaacg gtttaacagc caatgaatca 480 gggaggctaa tagatctcct caaagacgtg atggaatcaa tggataagga agagatggaa 540 ataacaacac attttcagag aaagagaaga gtgagggaca acatgaccaa gaaaatggtc 600 acacaaagaa caatagggaa gaaaaagcaa agactgaaca aaaggagcta tctaataaga 660 gcactgacac tgaacacaat gacaaaagat gcagaaagag gcaaattgaa aaggcgggca 720 attgcaacac ctggaatgca aatcagagga ttcgtgtacg ttgttgaaac actggcaagg 780 agtatctgtg agaaacttga gcaatctggg ctcccagttg gagggaatga aaagaaggcc 840 aaattggcaa atgttgtaag aaaaatgatg actaactcac aagatactga gctttccttt 900 acaattactg gagacaacac caaatggaat gagaatcaga atcctcggat gtttctagca 960 atgataacat atatcacaag gaaccaacct gaatggttta gaaatgtctt aagcattgcc 1020 cctataatgt tctcaaacaa aatggcgagg ttagggaaag ggtacatgtt cgaaagtaag 1080 aacatgaagc tacggacaca aataccggca gaaatgcttg caaacattga cctgaagtac 1140 tttaatgaat cgacaagaaa gaaaatcgag aaaataagac ctctactaat agatggcaca 1200 gccgcattga gtcctggaat gatgatgggc atgttcaaca tgcttagtac agtattagga 1260 gtctcaatcc taaatcttgg acagaagagg tacactaaaa ccacatattg gtgggacggg 1320 ctccaatcct ctgatgattt cgctctcata gtgaatgcac tggatcatga gggaatacaa 1380 gcaggagtag ataggttcta tagaacttgc aaattggttg ggatcaatat gagcaaaaag 1440 aagtcttaca taaatcgaac aggaacattt gaattcacta gcttttttta tcgctatgga 1500 tttgtagcca actttagtat ggagctaccc agctttggag tgtctgggat taacgaatcg 1560 gctgacatga gcattggtgt tacagtgata aagaacaaca tgataaacaa tgaccttgga 1620 ccagcaacag ctcaaatggc ccttcagcta ttcatcaagg actacaggta cacatacaga 1680 tgccataggg gtgatacaca aatccaaacg aggagatcat tcgagctgaa gaagctgtgg 1740 gagcagaccc gttcaaaggc aggactgttg gtttcggatg gaggaccaaa cctatacaat 1800 atccggaatc tccacattcc agaggtctgc ttgaaatggg aattgatgga tgaagattat 1860 caaggcaggc tgtgtaatcc tctgaatccg tttgtcagtc ataaggaaat tgagtccgtc 1920 aacaatgctg tagtaatgcc agctcatggt ccagccaaga gcatggaata tgatgctgtt 1980 gcaactacac attcatggat ccctaagagg aaccgttcca tcctcaacac cagtcaaagg 2040 ggaattcttg aagatgaaca gatgtaccag aagtgctgca atctatttga gaagttcttc 2100 cctagcagtt catatcgaag gccagttgga atttccagca tggtggaggc catggtatct 2160 agggcccgaa ttgatgcacg aattgacttc gagtctggaa ggattaagaa agaagagttt 2220 gctgagatca tgaagacctg ttccaccatt gaagagctca gacggcaaaa atag 2274 <210> 4 <211> 757 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PB1 protein of 01310 <400> 4 Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val Pro Ala Gln Asn   1 5 10 15 Ala Ile Ser Thr Thr Phe Pro Tyr Thr Gly Asp Pro Pro Tyr Ser His              20 25 30 Gly Thr Gly Thr Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln          35 40 45 Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu Thr Gly Ala Pro      50 55 60 Gln Leu Asn Pro Ile Asp Gly Pro Leu Pro Glu Asp Asn Glu Pro Ser  65 70 75 80 Gly Tyr Ala Gln Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu                  85 90 95 Glu Ser His Gly Ile Phe Glu Asn Ser Cys Leu Glu Thr Met Glu             100 105 110 Val Val Gln Gln Thr Arg Val Asp Lys Leu Thr Gln Gly Arg Gln Thr         115 120 125 Tyr Asp Trp Thr Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Thr     130 135 140 Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr Ala Asn Glu Ser 145 150 155 160 Gly Arg Leu Ile Asp Leu Leu Lys Asp Val Met Glu Ser Met Asp Lys                 165 170 175 Glu Glu Met Glu Ile Thr Thr His Phe Gln Arg Lys Arg Arg Val Val             180 185 190 Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr Ile Gly Lys Lys         195 200 205 Lys Gln Arg Leu Asn Lys Arg Ser Tyr Leu Ile Arg Ala Leu Thr Leu     210 215 220 Asn Thr Met Thr Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 225 230 235 240 Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val Tyr Val Val Glu                 245 250 255 Thr Leu Ala Arg Ser Ile Cys Glu Lys Leu Glu Gln Ser Gly Leu Pro             260 265 270 Val Gly Gly Asn Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys         275 280 285 Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe Thr Ile Thr Gly     290 295 300 Asp Asn Thr Lys Trp Asn Glu Asn Gln Asn Pro Arg Met Phe Leu Ala 305 310 315 320 Met Ile Thr Tyr Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val                 325 330 335 Leu Ser Ile Pro Ile Met Phe Ser Asn Lys Met Ala Arg Leu Gly             340 345 350 Lys Gly Tyr Met Phe Glu Ser Lys Asn Met Lys Leu Arg Thr Gln Ile         355 360 365 Pro Ala Glu Met Leu Ala Asn Ile Asp Leu Lys Tyr Phe Asn Glu Ser     370 375 380 Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu Ile Asp Gly Thr 385 390 395 400 Ala Ala Leu Ser Pro Gly Met Met Met Gly Met Phe Asn Met Leu Ser                 405 410 415 Thr Val Leu Gly Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr Thr             420 425 430 Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser Asp Asp Phe Ala         435 440 445 Leu Ile Val Asn Ala Leu Asp His Glu Gly Ile Gln Ala Gly Val Asp     450 455 460 Arg Phe Tyr Arg Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 465 470 475 480 Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe Thr Ser Phe Phe                 485 490 495 Tyr Arg Tyr Gly Phe Val Ala Asn Phe Ser Met Glu Leu Pro Ser Phe             500 505 510 Gly Val Ser Gly Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr         515 520 525 Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly Pro Ala Thr Ala     530 535 540 Gln Met Ala Leu Gln Leu Phe Ile Lys Asp Tyr Arg Tyr Thr Tyr Arg 545 550 555 560 Cys His Arg Gly Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu                 565 570 575 Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly Leu Leu Val Ser             580 585 590 Asp Gly Gly Pro Asn Leu Tyr Asn Ile Arg Asn Leu His Ile Pro Glu         595 600 605 Val Cys Leu Lys Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu     610 615 620 Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu Ile Glu Ser Val 625 630 635 640 Asn Asn Ala Val Val Met Pro Ala His Gly Pro Ala Lys Ser Met Glu                 645 650 655 Tyr Asp Ala Val Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg             660 665 670 Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu Asp Glu Gln Met         675 680 685 Tyr Gln Lys Cys Cys Asn Leu Phe Glu Lys Phe Phe Pro Ser Ser Ser     690 695 700 Tyr Arg Arg Pro Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 705 710 715 720 Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser Gly Arg Ile Lys                 725 730 735 Lys Glu Glu Phe Ala Glu Ile Met Lys Thr Cys Ser Thr Ile Glu Glu             740 745 750 Leu Arg Arg Gln Lys         755 <210> 5 <211> 2151 <212> DNA <213> Artificial Sequence <220> <223> PA gene of 01310 <400> 5 atggaagact ttgtgcgaca atgcttcaat ccaatgattg tcgagcttgc ggaaaaggca 60 atgaaagaat atggggaaga tccgaaaatc gaaactaata aatttgctgc aatatgcaca 120 cacttagaag tctgtttcat gtattctgat ttccatttta ttgatgagcg aggcgaatca 180 ataattgtag aatcgggcga cccaaatgca ttattgaaac accgatttga aataattgaa 240 gggagggatc gaacgatggc ctggacggtg gtgaatagta tatgcaatac tacaggagtc 300 gagaagccga aatttctccc agatttgtat gactacaagg agaatcgatt cattgaaatt 360 ggagtgacac ggagagaagt tcacatatac tatctagaga aagccaacaa aataaaatcc 420 gaaaagaccc acattcacat attctcattc actggggagg aaatggccac caaagcggac 480 tacacccttg atgaagaaag cagggcaagg atcaaaacca ggctgttcac tataaggcag 540 gaaatggctg gtaggggtct gtgggattcc tttcgtcagt ccgagagagg cgaagagaca 600 attgaagaga gatttgagat cactggaacc atgcgcaggc ttgccgacca aagcctccca 660 ccgaacttct ccagccttga aaactttaga gcctatgtgg atggattcga accgaacggc 720 tgcattgagg gcaagctttc tcaaatgtca aaagaagtga acgctagaat tgaaccattt 780 ctgagaacaa caccacgccc tattagatta cctgatgggc ctccctgttc ccaacgatca 840 aagttcttgc tgatggatgc acttaaatta agcatcgaag atccgagtca tgagggagag 900 gggataccgc tatatgatgc agtcaaatgc atgaagacat tttttggctg gaaagaaccc 960 aacattgtga aaccacatga aaagggcata aaccccaact acctcttggc ttggaagcag 1020 gtgttggcag aactccaaga tattgaacat gaagagaaaa ttccaaaaac aaagaacctg 1080 aagaaaacaa gccagttgaa gtgggcactt ggtgagaaca tggcaccaga gaaagtagac 1140 tttgaggact gtaaagatat tagcgatcta aaacaatatg acagtgatga accagagtct 1200 agatctctag caagctggat ccagaatgaa ttcaacaagg catgcgaatt gacagactca 1260 agttggattg aacttgatga aataggagaa gacgttgccc caattgagca cattgcaagt 1320 atgagaagga attacttcac agcagaagta tctcactgca gagctactga atacataatg 1380 aagggagtgt acataaacac agctttattg aatgcatcct gtgcagccat ggatgacttt 1440 caactgattc caatgataag caaatgcaga actaaagaag gaagacggaa aactaatcta 1500 tatggattca tcataaaggg aagatcccat ttgagaaatg acactgatgt agtaaacttt 1560 gtgagtatgg aattttctct tactgacccg aggctggagc cacacaaatg ggaaaagtac 1620 tgcgttctcg agataggaga catgctccta cggactgcaa taggccaagt gtcgaggccc 1680 atgtttctgt atgtgcggac caatggaacc tccaagatta agatgaaatg gggcatggaa 1740 atgaggcggt gccttcttca atccctccag caaattgaga gcatgattga ggccgagtct 1800 tctatcaagg agaaggacat gaccaaagaa ttctttgaaa acaaatcaga aacatggcca 1860 cggacagt cttgcaaaat ctgtgttcaa cagtctatat gcatctccac aactcgaggg gttttcagct 1980 gagtcaagaa aattacttct cattgttcag gcacttaggg acaacctgga acctgggacc 2040 ttcgatcttg gagggctata tgaagcaatt gaggagtgcc tgattaatga tccctgggtt 2100 ttgcttaatg catcttggtt caactccttc ctcacacatg cactgaaata g 2151 <210> 6 <211> 716 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PA protein of 01310 <400> 6 Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met Ile Val Glu Leu   1 5 10 15 Ala Glu Lys Ala Met Lys Glu Tyr Gly Glu Asp Pro Lys Ile Glu Thr              20 25 30 Asn Lys Phe Ala Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr          35 40 45 Ser Asp Phe His Phe Ile Asp Glu Arg Gly Glu Ser Ile Ile Val Glu      50 55 60 Ser Gly Asp Pro Asn Ala Leu Leu Lys His Arg Phe Glu Ile Ile Glu  65 70 75 80 Gly Arg Asp Arg Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn                  85 90 95 Thr Thr Gly Val Glu Lys Pro Lys Phe Leu Pro Asp Leu Tyr Asp Tyr             100 105 110 Lys Glu Asn Arg Phe Ile Glu Ile Gly Val Thr Arg Arg Glu Val His         115 120 125 Ile Tyr Tyr Leu Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His     130 135 140 Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala Thr Lys Ala Asp 145 150 155 160 Tyr Thr Leu Asp Glu Glu Ser Arg Ala Arg Ile Lys Thr Arg Leu Phe                 165 170 175 Thr Ile Arg Gln Glu Met Ala Gly Arg Gly Leu Trp Asp Ser Phe Arg             180 185 190 Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg Phe Glu Ile Thr         195 200 205 Gly Thr Met Arg Arg Leu Ala Asp Gln Ser Leu Pro Pro Asn Phe Ser     210 215 220 Ser Leu Glu Asn Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly 225 230 235 240 Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu Val Asn Ala Arg                 245 250 255 Ile Glu Pro Phe Leu Arg Thr Thr Pro Arg Pro Ile Arg Leu Pro Asp             260 265 270 Gly Pro Pro Cys Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu         275 280 285 Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu Gly Ile Pro Leu     290 295 300 Tyr Asp Ala Val Lys Cys Met Lys Thr Phe Phe Gly Trp Lys Glu Pro 305 310 315 320 Asn Ile Val Lys Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu                 325 330 335 Ala Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile Glu His Glu Glu             340 345 350 Lys Ile Pro Lys Thr Lys Asn Leu Lys Lys Thr Ser Gln Leu Lys Trp         355 360 365 Ala Leu Gly Glu Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys     370 375 380 Lys Asp Ile Ser Asp Leu Lys Gln Tyr Asp Ser Asp Glu Pro Glu Ser 385 390 395 400 Arg Ser Leu Ala Ser Trp Ile Gln Asn Glu Phe Asn Lys Ala Cys Glu                 405 410 415 Leu Thr Asp Ser Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val             420 425 430 Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn Tyr Phe Thr Ala         435 440 445 Glu Val Ser His Cys Arg Ala Thr Glu Tyr Ile Met Lys Gly Val Tyr     450 455 460 Ile Asn Thr Ala Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe 465 470 475 480 Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys Glu Gly Arg Arg                 485 490 495 Lys Thr Asn Leu Tyr Gly Phe Ile Ile Lys Gly Arg Ser His Leu Arg             500 505 510 Asn Asp Thr Asp Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr         515 520 525 Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr Cys Val Leu Glu     530 535 540 Ile Gly Asp Met Leu Leu Arg Thr Ala Ile Gly Gln Val Ser Arg Pro 545 550 555 560 Met Phe Leu Tyr Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys                 565 570 575 Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser Leu Gln Gln Ile             580 585 590 Glu Ser Met Ile Glu Ala Glu Ser Ser Ile Lys Glu Lys Asp Met Thr         595 600 605 Lys Glu Phe Phe Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser     610 615 620 Pro Lys Gly Val Glu Glu Gly Ser Ile Gly Lys Val Cys Arg Thr Leu 625 630 635 640 Leu Ala Lys Ser Val Phe Asn Ser Leu Tyr Ala Ser Pro Gln Leu Glu                 645 650 655 Gly Phe Ser Ala Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu             660 665 670 Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly Gly Leu Tyr Glu         675 680 685 Ala Ile Glu Glu Cys Leu Ile Asn Asp Pro Trp Val Leu Leu Asn Ala     690 695 700 Ser Trp Phe Asn Ser Phe Leu Thr His Ala Leu Lys 705 710 715 <210> 7 <211> 2280 <212> DNA <213> Artificial Sequence <220> <223> PB2 gene of KBNP-0028 <400> 7 atggagagaa taaaagaact aagagatttg atgtcgcagt ctcgcactcg cgagatacta 60 acaaaaacca ctgtggacca tatggccata atcaggaaat acacatcagg aagacaggag 120 aagaatcctg ctctcagaat gaaatggatg atggcaatga agtatccgat tacagctgac 180 agaagaataa tggagatgat ccctgaaaga aatgagcaag gtcaaactct ttggagcaaa 240 acaaatgacg ctggatcaga cagagtgatg gtatcacctc tggcagtaac gtggtggaac 300 aggaatggac caacgacaag tacagtccat tatccaaaag tatataaaac ctactttgaa 360 aaggtagaaa ggttgaaaca cggaactttt ggccccgtcc atttccggaa tcaagtcaaa 420 atacgtcgca gggttgacat aaacccgggc catgctgatc tcagtgctaa agaagcacaa 480 gatgtcataa tggaggtggt cttcccaaac gaggttgggg ccaggatatt gacatcagaa 540 tcgcaattga caataacaaa agaaaagaag gagaaacttc aggactgcaa gattgctcct 600 ttaatggtgg catatatgtt ggaaagagaa ctggttcgca agaccagatt cctaccagtg 660 gccggtggga caagcagcgt atacattgag gtattgcatt tgactcaagg aacctgctgg 720 gaacaaatgt acaccccggg aggggaggtg agaaatgatg atgttgacca gagtttaatc 780 attgctgcta ggaacatcgt taggagggca acagtatcag cagacccatt agcttcgctt 840 ttggagatgt gccatagtac acaaattggc gggataagaa tggtggacat ccttagacag 900 aacccaacag aagagcaagc tgtggacata tgcaaggcag caatgggtct aaggatcagt 960 tcatccttca gctttggggg tttcactttc aaaagaacaa gtgggtcatc aatcaaaaga 1020 gaggaagaag tgcttacagg caacctccag acattgaaaa taaggatata tgaaggatat 1080 gaggaattca caatggttgg aagaagagca acagccatcc taagaagagc aaccagaagg 1140 ctgatccaac tgatagttag tgggagagac gaacaatcaa tcgctgaagc aatcatcgta 1200 gctatggtgt tctcacaaga ggattgcatg ataaaggcag tccgaggtga tttgaatttc 1260 gtgaacagag cgaaccaacg gctgaatccc atgcaccaac ttcttaggca cttccaaaag 1320 gatgcaaaag tattgtttca aaactgggga gttgaaccca ttgacaatgt catggggatg 1380 attgggatat tgcctgacat gacccccagc acggaaatgt cactgagagg ggtgagagtc 1440 agtaaaatgg gagtggacga atattccagc accgagagag tggtcgtaag cattgatcgt 1500 ttcttaagag tccgagatca aaggggaaac atactcctat cccctgaaga ggtcagtgaa 1560 acgcaaggaa ctgaaaagct gacgataaca tactcatcgt ctatgatgtg ggagatcaac 1620 ggtccggaat cagtgctggt taatacatac caatggatca ttagaaattg ggagaccgtg 1680 aagattcaat ggtcccaaga ccccacaatg ctatacaata agatggagtt tgaacccttt 1740 caatctttgg tacctaaagc tgccagaggc caatatagtg gatttgtgag aacgctattc 1800 cagcaaatgc gtgatgtgtt ggggacattt gacactgtcc aaataataa gctactacca 1860 tttgcagctg cccctccaga gcagagtagg atgcagtttt cttctctaac tgtgaatgtg 1920 aggggttcag gaatgagaat acttgtgaga ggtaactccc ctgtgtttaa ctacaacaag 1980 gcaaccaaga ggcttacagt ccttggaaaa gatgcaggtg gacttgcaga agacccagat 2040 gagggaacgg caggggtgga atcagcagta ttgagaggat ttctaatttt aggtaaagaa 2100 gacaaaagat atggaccagc attgagcatc aacgaattga gcaatcttgc gaaaggggag 2160 agagctaatg ttttgatagg gcaaggagac atagtgttgg tgatgaaacg gaaacgggac 2220 tctagcatac ttactgacag ccagacagcg accaaaagaa ttcggacggc catcaattag 2280                                                                         2280 <210> 8 <211> 759 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of PB2 protein of KBNP-0028 <400> 8 Met Glu Arg Ile Lys Glu Leu Arg Asp Leu Met Ser Gln Ser Arg Thr   1 5 10 15 Arg Glu Ile Leu Thr Lys Thr Thr Val Asp His Met Ala Ile Ile Arg              20 25 30 Lys Tyr Thr Ser Gly Arg Gln Glu Lys Asn Pro Ala Leu Arg Met Lys          35 40 45 Trp Met Met Ala Met Lys Tyr Pro Ile Thr Ala Asp Arg Arg Ile Met      50 55 60 Glu Met Ile Pro Glu Arg Asn Glu Gln Gly Gln Thr Leu Trp Ser Lys  65 70 75 80 Thr Asn Ala Gly Ser Asp Arg Val Met Val Ser Pro Leu Ala Val                  85 90 95 Thr Trp Trp Asn Arg Asn Gly Pro Thr Thr Ser Thr Val His Tyr Pro             100 105 110 Lys Val Tyr Lys Thr Tyr Phe Glu Lys Val Glu Arg Leu Lys His Gly         115 120 125 Thr Phe Gly Pro Val His Phe Arg Asn Gln Val Lys Ile Arg Arg Arg     130 135 140 Val Asp Ile Asn Pro Gly His Ala Asp Leu Ser Ala Lys Glu Ala Gln 145 150 155 160 Asp Val Ile Met Glu Val Val Phe Pro Asn Glu Val Gly Ala Arg Ile                 165 170 175 Leu Thr Ser Glu Ser Gln Leu Thr Ile Thr Lys Glu Lys Lys Glu Lys             180 185 190 Leu Gln Asp Cys Lys Ile Ala Pro Leu Met Val Ala Tyr Met Leu Glu         195 200 205 Arg Glu Leu Val Arg Lys Thr Arg Phe Leu Pro Val Ala Gly Gly Thr     210 215 220 Ser Ser Val Tyr Ile Glu Val Leu His Leu Thr Gln Gly Thr Cys Trp 225 230 235 240 Glu Gln Met Tyr Thr Pro Gly Gly Glu Val Arg Asn Asp Asp Val Asp                 245 250 255 Gln Ser Leu Ile Ile Ala Ala Arg Asn Ile Val Arg Arg Ala Thr Val             260 265 270 Ser Ala Asp Pro Leu Ala Ser Leu Leu Glu Met Cys His Ser Thr Gln         275 280 285 Ile Gly Gly Ile Arg Met Val Asp Ile Leu Arg Gln Asn Pro Thr Glu     290 295 300 Glu Gln Ala Val Asp Ile Cys Lys Ala Ala Met Gly Leu Arg Ile Ser 305 310 315 320 Ser Ser Phe Ser Phe Gly Gly Phe Thr Phe Lys Arg Thr Ser Gly Ser                 325 330 335 Ser Ile Lys Arg Glu Glu Glu Val Leu Thr Gly Asn Leu Gln Thr Leu             340 345 350 Lys Ile Arg Ile Tyr Glu Gly Tyr Glu Glu Phe Thr Met Val Gly Arg         355 360 365 Arg Ala Thr Ala Ile Leu Arg Arg Ala Thr Arg Arg Leu Ile Gln Leu     370 375 380 Ile Val Ser Gly Arg Asp Glu Gln Ser Ile Ala Glu Ala Ile Ile Val 385 390 395 400 Ala Met Val Phe Ser Gln Glu Asp Cys Met Ile Lys Ala Val Arg Gly                 405 410 415 Asp Leu Asn Phe Val Asn Arg Ala Asn Gln Arg Leu Asn Pro Met His             420 425 430 Gln Leu Leu Arg His Phe Gln Lys Asp Ala Lys Val Leu Phe Gln Asn         435 440 445 Trp Gly Val Glu Pro Ile Asp Asn Val Met Gly Met Ile Gly Ile Leu     450 455 460 Pro Asp Met Thr Pro Ser Thr Glu Met Ser Leu Arg Gly Val Arg Val 465 470 475 480 Ser Lys Met Gly Val Asp Glu Tyr Ser Ser Thr Glu Arg Val Val Val                 485 490 495 Ser Ile Asp Arg Phe Leu Arg Val Arg Asp Gln Arg Gly Asn Ile Leu             500 505 510 Leu Ser Pro Glu Glu Val Ser Glu Thr Gln Gly Thr Glu Lys Leu Thr         515 520 525 Ile Thr Tyr Ser Ser Ser Met Met Trp Glu Ile Asn Gly Pro Glu Ser     530 535 540 Val Leu Val Asn Thr Tyr Gln Trp Ile Ile Arg Asn Trp Glu Thr Val 545 550 555 560 Lys Ile Gln Trp Ser Gln Asp Pro Thr Met Leu Tyr Asn Lys Met Glu                 565 570 575 Phe Glu Pro Phe Gln Ser Leu Val Pro Lys Ala Ala Arg Gly Gln Tyr             580 585 590 Ser Gly Phe Val Arg Thr Leu Phe Gln Gln Met Arg Asp Val Leu Gly         595 600 605 Thr Phe Asp Thr Val Gln Ile Ile Lys Leu Leu Pro Phe Ala Ala Ala     610 615 620 Pro Pro Glu Gln Ser Arg Met Gln Phe Ser Ser Leu Thr Val Asn Val 625 630 635 640 Arg Gly Ser Gly Met Arg Ile Leu Val Arg Gly Asn Ser Pro Val Phe                 645 650 655 Asn Tyr Asn Lys Ala Thr Lys Arg Leu Thr Val Leu Gly Lys Asp Ala             660 665 670 Gly Gly Leu Ala Glu Asp Pro Asp Glu Gly Thr Ala Gly Val Glu Ser         675 680 685 Ala Val Leu Arg Gly Phe Leu Ile Leu Gly Lys Glu Asp Lys Arg Tyr     690 695 700 Gly Pro Ala Leu Ser Ile Asn Glu Leu Ser Asn Leu Ala Lys Gly Glu 705 710 715 720 Arg Ala Asn Val Leu Ile Gly Gln Gly Asp Ile Val Leu Val Met Lys                 725 730 735 Arg Lys Arg Asp Ser Ser Ile Leu Thr Asp Ser Gln Thr Ala Thr Lys             740 745 750 Arg Ile Arg Thr Ala Ile Asn         755 <210> 9 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> M13F primer <400> 9 gtaaaacgac ggccag 16 <210> 10 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> M13R primer <400> 10 caggaaacag ctatgac 17

Claims (21)

PB2 protein coding polynucleotide of low pathogenic avian influenza virus as an active ingredient
Attenuated embryonated egg composition for producing a high-proliferative H1N1 strain avian influenza virus.
The method of claim 1,
Wherein the PB2 protein of the low pathogenic avian influenza virus comprises one or more of the following characteristics, attenuated embryonated egg hyperproliferative H1N1 strain avian influenza virus production composition:
Wherein the amino acid 66 is isoleucine (I),
The amino acid 88 is lysine (K),
When the amino acid 109 is isoleucine (I),
When the amino acid 133 is isoleucine (I),
When the amino acid 157 is arginine (R),
Amino acid 286 is glycine (G),
When the amino acid 315 is isoleucine (I),
373 amino acid is leucine (L)
The amino acid at position 451 is isoleucine (I),
The amino acid 575 is valine (V), and
The amino acid 674 is alanine (A).
According to claim 2, wherein the low pathogenic avian influenza A / chicken / Korea / 01310/2001 (H9N2) strain, attenuated embryonated egg hyperproliferative H1N1 strain avian influenza virus production composition.
According to claim 3, wherein the low pathogenic avian influenza PB2 protein is a protein consisting of the amino acid sequence of SEQ ID NO: 2, attenuated embryonated egg hyperproliferative H1N1 strain avian influenza virus production composition.
The composition of any one of claims 1 to 4, wherein the H1N1 strain avian influenza virus is an A / Puerto Rico / 8/34 (H1N1) virus, and attenuated embryonated egg high-proliferative H1N1 strain avian influenza virus.
Polymerase B1 (PB1) coding polynucleotides, polymerase A (PA) coding polynucleotides, hemagglutinin protein (HA) coding polynucleotides, nucleocapsid (NP) coding polynucleotides, neuramidase of H1N1 strain avian influenza virus (NA) coding polynucleotides, matrix protein (M) coding polynucleotides, and nonstructural protein (NS) coding polynucleotides; And
PB2 Protein Coding Polynucleotides of Low Pathogenic Avian Influenza Viruses
Comprising a recombinant expression vector.
The method according to claim 6,
The PB2 protein of the low pathogenic avian influenza virus comprises at least one of the following characteristics, recombinant expression vector:
Wherein the amino acid 66 is isoleucine (I),
The amino acid 88 is lysine (K),
When the amino acid 109 is isoleucine (I),
When the amino acid 133 is isoleucine (I),
When the amino acid 157 is arginine (R),
Amino acid 286 is glycine (G),
When the amino acid 315 is isoleucine (I),
373 amino acid is leucine (L)
The amino acid at position 451 is isoleucine (I),
The amino acid 575 is valine (V), and
The amino acid 674 is alanine (A).
The recombinant expression vector according to claim 7, wherein the low pathogenic avian influenza is A / chicken / Korea / 01310/2001 (H9N2) strain.
The recombinant expression vector of claim 8, wherein the PB2 protein of the low pathogenic avian influenza is a PB2 protein consisting of the amino acid sequence of SEQ ID NO: 2. 10.
The recombinant expression vector of claim 6, wherein the H1N1 strain avian influenza virus is an A / Puerto Rico / 8/34 (H1N1) virus.
10. A recombinant influenza virus wherein the H1N1 strain avian influenza virus is transformed with the recombinant expression vector of any one of claims 6-9.
The recombinant influenza virus of claim 11, wherein the H1N1 strain avian influenza virus is an A / Puerto Rico / 8/34 (H1N1) virus.
The recombinant influenza virus of claim 12, wherein the PB2 protein coding polynucleotide is a coding polynucleotide of SEQ ID NO: 1.
An avian influenza virus vaccine containing a recombinant influenza virus transformed with an H1N1 strain avian influenza virus as the recombinant expression vector of any one of claims 6 to 9.
The vaccine of claim 14, wherein the H1N1 strain avian influenza virus is an A / Puerto Rico / 8/34 (H1N1) virus.
The vaccine of claim 15, wherein the PB2 protein coding polynucleotide contains a recombinant influenza virus that is the coding polynucleotide of SEQ ID NO: 1.
16. The vaccine according to claim 15, wherein the vaccine is a sadox vaccine or a live monkey.
10. The composition for diagnosing avian influenza virus comprising an antiserum against a recombinant influenza virus transformed with the recombinant expression vector of claim 6 as an active ingredient.
The composition for diagnosing avian influenza virus according to claim 18, wherein the recombinant influenza virus is a PB2 protein encoding polynucleotide encoding a polynucleotide of SEQ ID NO: 1.
The composition for diagnosing avian influenza virus of claim 18, wherein the antiserum is obtained from a bird or a mammal.
Recombinant A / Puerto Rico / 8/34 (H1N1) virus of accession number KCTC 12076BP.

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