KR20180046968A - Virus like particle vaccine for h5 influenza virus and the method for the same - Google Patents

Abstract

Disclosed is virus-like particle vaccine, which comprises a hemagglutinin (HA) protein and a neuraminidase (NA) protein derived from H5N8, and comprises an M1 protein derived from H1N1. The virus-like particle vaccine can induce an immune response not only by intramuscular injection but also by respiratory injection.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a vaccine for H5 avian influenza virus-like particles,

The present invention relates to an H5 avian influenza virus-like particle vaccine and a method for producing the same.

Influenza virus belongs to Orthomyxoviridae and is divided into A type, B type and C type. As a virus having eight single-stranded RNAs as a genome, Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP) , Matrix proteins 1 and 2 (Matrix, M1 and M2), polymerase units A, B1 and B2 (Polymerase subunit A, B1 and B2 respectively PA and PB1 and PB2) and nonstructural proteins 1 and 2 &2; NS1 and NS2, respectively) are created.

Influenza A virus subtype H5N8 is a subtype of influenza A that started in 1983 in a country that has become depressed in Ireland and has high fever and diarrhea and has been exposed to dead poultry (turkeys, chickens, ducks ). H5N8 was detected in a chicken in a live chicken market during a survey of chicken and goose infected birds in China in 2010, when there was no report for 27 years. It is known to be highly pathogenic in poultry including ducks when the symptoms confirmed outdoors are considered. In the case of H5N8 strain, the H5N1 strain and the serotype were different in the past. However, the infection symptom and the virulence of the H5N8 strain are not so different from those of the H5N1 strain.

The H1N1 influenza virus is the first type of influenza A virus found in humans in the spring of 2009, and among the genetic traits of H1N1 influenza in 2009, HA is the same as the one that was isolated from swine influenza in 1999 in the United States. However, NA and M are similar to those isolated from avian influenza in Europe. Six genes from swine flu, bird flu and human influenza are mixed in the American swine influenza. These gene mixtures were not found until they were transmitted from pigs to humans. This propagation path was unusual in light of previous influenza transmission between pigs in the United States. H1N1 influenza has not been experienced by most humans, so the susceptibility of H1N1 influenza is high and the pandemic has been fully anticipated.

Commonly used vaccine formulations, such as attenuated live vaccines or inactivated vaccines, are effective, but safety issues have arisen, such as the toxicity of an attenuated or inactivated virus. In addition, it takes a long time because it requires subculture several times during the attenuation process. Therefore, the vaccine formulation to replace it is a subunit vaccine or a recombinant protein vaccine. The subunit vaccine does not inoculate the entire pathogen. The recombinant protein vaccine is prepared by expressing the recombinant protein as a recombinant protein using an antigen, such as Escherichia coli or animal cells, and then using the recombinant protein as a vaccine to be. Subunit vaccines or recombinant protein vaccines are more safe than live vaccines or inactivated vaccines because they only inoculate the antigenic site, not the entire pathogen, but are less efficient than the vaccine. In order to overcome this, several inoculations or immune supplements are mixed.

Virus Like Particle means a recombinant protein having a virus-like shape. Virus-like particles are assembled into a virus-like form through binding of virus structural proteins, but the virus gene is not included in the virus-like particle during the assembly process. The virus-like particles are very similar to actual viruses, so they exhibit high immunogenicity when injected into the body, but do not contain the viral gene. Therefore, they act as safe antigens that can not multiply in the body. That is, it is structurally similar to virus because it is composed only of structural protein of virus, but it is a particle that can not replicate or replicate in cell because there is no gene. In this case, it shows a better defense mechanism against the pathogen by inducing a better immune response than the virus containing only the toxic protein. It shows defense mechanism against the pathogen even when it is inoculated more than the subunit vaccine which needs to be inoculated several times. Activity. Based on these advantages, vaccines against various pathogens using virus-like particles have been developed very actively, and some virus-like particle vaccines have been commercialized.

The advantages of the virus-like particle vaccine are as follows. The surface structure is repeatedly constituted and has unity. It is presented to the immune cells in the body while maintaining the natural virus antigen structure, and has an advantage of being able to efficiently induce the immune response. It is also considered to be one of the greatest advantages because it does not contain the viral gene necessary for the viral propagation and therefore it is impossible to infect animals and is very safe. In general, recombinant vaccines are unstable due to environmental conditions because they express only specific antigen proteins and are stored in solution. However, since virus-like particles have a particle structure similar to viruses, their structure changes easily due to environmental conditions There is also some research that has stability. In addition, since it forms a particle structure, antigens derived from one kind of pathogen, as well as other viruses and antigens derived from pathogens such as bacteria, are simultaneously expressed in virus-like particles, so that they can simultaneously protect against various infectious diseases by a single vaccination There is also an advantage.

It is estimated that the avian influenza has a low utility value for vaccination due to its variety of serotypes and its good mutation. In other words, vaccines must be made in the same serotype for each viral serotype so that they can have a proper preventive effect. The low-pathogenic H9N2 virus has already been commercialized as a vaccine and has been used in many poultry farms and has been highly evaluated for its efficacy. However, there is no protective effect on other serotypes and there is a limited effect on the same serotype H9N2.

Prophylactic vaccines can also be made against H5N1 or H5N8, which are highly pathogenic. However, the use of the H5N1 virus for vaccination does not prevent the H5N8 strain because the serotype structure is different. Moreover, in the case of avian influenza vaccine, it is inevitable that there will be a lot of risk when it is manufactured as a virulent vaccine. The use of raw monocotyledons may result in a combination of vaccine and open-stock or a genetic mutation that can lead to the emergence of a new HPAI virus that can not be controlled. Therefore, it is the correct direction that vaccination against HPAI virus is absolutely prohibited, not to mention low-pathogenic influenza (LPAI).

Therefore, it is necessary to prepare each vaccine for a specific virus serotype and a vaccine against H5N8 newly emerging as described above is necessary. In addition, there is a clear limit to the natural monologue against avian influenza.

The present invention is directed to providing virus-like particles as a vaccine against H5 avian influenza.

As a technical means for achieving the above-mentioned technical object, a virus-like particle vaccine according to an aspect of the present invention comprises hemagglutinin (HA) protein and neuraminidase (NA) protein derived from H5N8 virus, RTI ID = 0.0 > M1 < / RTI > protein from the H1N1 virus.

The H5N8 virus may be A / mallard / Korea / W452 / 2014 (H5N8).

The H1N1 virus may be A / Puerto Rico / 8-V24 / 1934 (H1N1).

The hemagglutinin (HA) protein may be defined as the amino acid sequence of SEQ ID NO: 2.

The neuraminidase (NA) protein may be defined as the amino acid sequence of SEQ ID NO: 4.

The M1 protein may be defined as the amino acid sequence of SEQ ID NO: 6.

A vaccine composition according to another aspect of the present invention comprises virus-like particles as an active ingredient.

The method for preventing avian influenza according to another aspect of the present invention may include the step of administering the vaccine composition to algae.

A method for producing a virus-like particle according to another aspect of the present invention includes the steps of preparing a recombinant vector comprising a gene of a hemagglutinin (HA) protein derived from a H5N8 virus, Transposing the recombinant plasmid to a Bacmid expressing a recombinant baculovirus (NA) protein, transfecting the recombinant Bacmid into an insect cell, obtaining a recombinant baculovirus from the insect cell, contacting the recombinant baculovirus and H1N1 (HA) protein, the neuraminidase (NA) protein, and the M1 protein in the insect cells are infected with a bacterium that expresses the M1 protein derived from the virus, And separating the virus-like particles forming the envelope.

According to one embodiment of the present invention, a vaccine of A / mallard / Korea / W452 / 2014 (H5N8), which is not existed before, especially virus-like particle vaccine, shows its function and activity sufficiently as a vaccine. In addition, according to another embodiment of the present invention, the vaccine effect of the virus-like particle vaccine can be seen not only through intramuscular injection but also through respiratory injection, especially when re-vaccinated.

FIG. 1 is a graph illustrating western blot data for confirming VLP formation according to an embodiment of the present invention.
FIG. 2 is a graph showing an animal inoculation and immune response ELISA assay data for measuring the activity of VLPs according to an embodiment of the present invention
Figure 3 shows the DNA and amino acid sequence of the HA protein according to one embodiment of the present invention
Figure 4 shows the DNA and amino acid sequence of the NA protein according to one embodiment of the present invention
Figure 5 shows the DNA and amino acid sequence of the M1 protein according to one embodiment of the present invention

Hereinafter, the present invention will be described in more detail. However, it should be understood that the present invention is not limited thereto, and the present invention is only defined by the scope of the following claims.

The present invention relates to an HA protein [GenBank: KJ746111.1] and an NA protein [GenBank: KJ746113.1] derived from A / mallard / Korea / W452 / 2014 (H5N8) avian influenza virus, and A / Puerto Rico / (VLPs) comprising the M1 protein [GenBank: CY084183.1] derived from / 1934 (H1N1).

The virus-like particle (VLP) means a kind of antigen having a virus-like shape. Virus-like particles are assembled into a virus-like form through binding of virus structural proteins, but the virus gene is not included in the virus-like particle during the assembly process.

The hemagglutinin (HA) refers to a substance capable of aggregating red blood cells. Typically, hemagglutinin refers to an antigenic protuberance that protrudes from the envelope of a virus, and more specifically refers to a glycoprotein found in the envelope of influenza virus. The antigenic lobes of the influenza virus envelope include hemagglutinin and neuraminidase (NA), where the hemagglutinin serves to attach the virus to parasitic and proliferative host cells, Laminidase is known to be used for the end of parasitic and proliferative and deviating from host cells. Hemagglutinin is composed of a form including a head or stem domain protruding out of the membrane, a transmembrane domain permeating the membrane, and a cytoplasmic tail inside the cytoplasm.

According to a preferred embodiment of the present invention, the hemagglutinin (HA) protein may be derived from H5N8 influenza virus.

The entire sequence of the above-described hemagglutinin (HA) protein can be defined, for example, as the amino acid sequence of SEQ ID NO: 2.

The above hemagglutinin (HA) protein as well as SEQ ID NO: 2, as well as those having a sequence of 70% or more, specifically 80% or more, more specifically 90% or more, still more specifically 95% or more, May comprise an amino acid sequence that exhibits 98% or more sequence homology.

Also, as the sequence showing such sequence homology, any mutant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added is also within the scope of the present invention, provided that the amino acid sequence exhibits substantially the same or a corresponding biological activity as the above proteins It is self-evident.

The neuraminidase (NA) protein is an enzyme of the glycoprotein present in the viral envelope membrane, which is necessary when the virus enters or exits the host cell. Specifically, the neuraminidase of the virus functions to cleave the terminal of neuraminic acid from the glycan structure of the infected cell surface. There are 9 subtypes (N1 ~ N9), and N1 and N2 are seen in humans.

According to a preferred embodiment of the present invention, the neuraminidase (NA) protein may be derived from A / mallard / Korea / W452 / 2014 (H5N8) virus.

The entire sequence of the neuraminidase (NA) protein can be defined, for example, as the amino acid sequence of SEQ ID NO: 4.

The above-mentioned neuraminidase (NA) protein has 70% or more, specifically 80% or more, more specifically 90% or more, more specifically 95% or more, most specifically, May contain an amino acid sequence that exhibits 98% or more sequence homology.

Also, as the sequence showing such sequence homology, any mutant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added is also within the scope of the present invention, provided that the amino acid sequence exhibits substantially the same or a corresponding biological activity as the above proteins It is self-evident.

The M1 protein is a matrix protein of influenza virus and forms a coating film inside the virus envelope. The M1 protein consists of two domains, each of which is linked by a linker sequence. The M1 protein binds to viral RNA, which is not specific to a particular RNA sequence, but is linked through an amino acid-rich peptide sequence.

According to a preferred embodiment of the present invention, the M1 protein may be derived from A / Puerto Rico / 8-V24 / 1934 (H1N1) virus.

The entire sequence of the M1 protein can also be defined, for example, as the amino acid sequence of SEQ ID NO: 6.

The M1 protein has not only the sequence of SEQ ID NO: 6 but also a sequence having at least 70%, specifically at least 80%, more specifically at least 90%, even more specifically at least 95%, most particularly at least 98% And may include an amino acid sequence that exhibits homology.

Also, as the sequence showing such sequence homology, any mutant having an amino acid sequence in which a part of the sequence is deleted, modified, substituted or added is also within the scope of the present invention, provided that the amino acid sequence exhibits substantially the same or a corresponding biological activity as the above proteins It is self-evident.

A vaccine composition according to another aspect of the present invention comprises the above-mentioned virus-like particles as an active ingredient.

In addition to the above virus-like particles, pharmaceutically acceptable carriers or other additives may be added to the vaccine composition, so that detailed description thereof will be omitted.

A method of producing virus-like particles according to another aspect of the present invention comprises the steps of: a) preparing a recombinant vector comprising a gene of hemagglutinin (HA) protein derived from A / mallard / Korea / W452 / 2014 (H5N8) B) transposing the recombinant plasmid to a Bacmid expressing a neuraminidase (NA) protein derived from A / mallard / Korea / W452 / 2014 (H5N8) virus; c) transfecting the recombinant Bacmid into insect cells E) obtaining a recombinant baculovirus from the insect cell; b) expressing the recombinant baculovirus and the M1 protein derived from the A / Puerto Rico / 8-V24 / 1934 (H1N1) virus; F) inserting the hemagglutinin (HA) protein, the neuraminidase (NA) protein and the M1 protein into particles to form an envelope in the insect cells; And a step of removing the virus-like particles.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example 1: Fabrication of VLPs containing influenza H5N8 virus HA NA antigen

A / mallard / Korea / W452 / 2014 (H5N8) The HA and NA specific primers are prepared from the gene of the virus and the insert is secured by PCR. The HA protein has the nucleotide sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2, and the NA protein has the nucleotide sequence of SEQ ID NO: 2 or the amino acid sequence of SEQ ID NO: 2. After securing the inserts, they were inserted into pFastBac1 using restriction enzymes BamH1 and Xho1, transformed into DH5a using heat shock method, and mini-prep was performed to obtain recombinant pFastBac1. Recombinant pFastBac1 was transformed into DH10Bac using the heatshock method, and the recombinant bacmid was obtained by midi-prep. The sequence was confirmed by marcrogen, a company specializing in sequencing.

 Recombinant Bacmid is transfected into sf9 cell using Lipfectamin agent and cultured at 27 ° C for 3 days to obtain recombinant Baculovirus. The obtained recombinant Baculovirus was cultured for 3 days in T75 flask and 100 ml culture, respectively, to obtain Baculovirus.

Then, in order to prepare VLPs, HA and NA of influenza H5N8 and recombinant Baculovirus expressing M1 having the nucleotide sequence of SEQ ID NO: 5 or M1 having the sequence of SEQ ID NO: 6 were added to a 400 ml sf9 cell culture at a concentration of 1 × 10 ⁶ cells / . After incubation for 3 days, centrifugation was performed at 4000 rpm for 20 minutes to obtain supernatant. The VLPs in the supernatant were concentrated to 1/10 volume using a 100KDa membrane, and a sucrose gradient purification method was used to prepare a 60% 20% sucrose layer. The concentrated sample was loaded on the VLP and centrifuged at 30000 rpm for 1 hour To isolate the layer between 60% and 20% to obtain influenza H5N8 VLP.

Test Example 1: VLP assay expressing HA of influenza A virus H5N8

The concentration of H5N8 VLP was measured by BCA assay using Pierce ™ BCA Protein Assay Kit23225. BSA standard and VLP sample of known concentration of 25μl were loaded on a 96-well plate, and then reagent A and reagent B were mixed at a ratio of 50: 1, 200ul per well, incubated at 37 ° C and absorbance at 560nm When the concentration was measured, a concentration of 0.6 mg / ml was confirmed.

 HA assay was performed to measure HA activity of H5N8 VLP. 50ul PBS was added to a V-bottom 96-well plate, followed by dilution of the VLP sample to half concentration sequentially with 50 μl of 0.5% RBC to form a spot. The result was 512 HAU / 50ul HA titer Respectively.

Western blot analysis was performed to confirm protein expression of VLP. After loading the sample in 12% acrylamide gel and running for 45 minutes at 200 V, the gel separated proteins were transferred to PVDF membrane using semi dry transfer method and blackened in 5% skim milk for 30 minutes. The ferat-derived antibody against H5N8 virus anti H5N8 virus pAb) was diluted 1: 500 in 1% skim milk condition at 4 ° C overnight, washed with PBST buffer (washing buffer) 3 times for 10 minutes, then incubated with goat anti-Ferat IgG antibody (goat anti ferat IgG HRP) was diluted 1: 5000 and detected and protein expression was confirmed. As a result, the hemagglutinin protein, which is the antigen protein of virus, appeared in proportion to the amount of gel loaded in 63 ~ 75KDa and was proportional to the concentration of M1 protein at 25KDa.

The western blot results are shown in FIG. 1, and each lane is as follows.

Lane1: size marker

Lane 2 to 5: influenza H5N8 VLP (influenza H5N8 virus-like particle)

Lane 6 to 8: influenza H5N8 inactivated virus (influenza H5N8 inactivated virus)

Test Example 2: Animal Inoculation and Immune Response Analysis of H5N8 VLP

 H5N8 VLP was injected intramuscularly and intranasal route in 6-week-old female BALB / C at 10 ug each. After the second injection with the same conditions, the same orbital blood was collected after 3 weeks and the serum was secured and the ELISA assay was performed to confirm the antibody formation. Inactivated virus at 4 ug / ml was coated on a 96-well plate at 4 ° C overnight. The plate was incubated at 37 ° C for 1 hour and 30 minutes with 3% BSA solution. After washing three times with PBST, the serum was diluted and 100 ul was added per well. Incubation was carried out at 37 ° C for 1 hour and 30 minutes. After 3 washes of PBST, the antibody against goat-derived mouse IgG or goat-derived mouse IgA was treated with 2-DAB for 1 hour at 37 ° C and then washed 6 times with PBST. After incubation at room temperature for 10 minutes, TMB solution was used to stop the color reaction with 0.5 M sulfuric acid solution and absorbance was measured at 450 nm wavelength.

The ELISA assay results are shown in FIG. 2. The graphs are as follows.

VLP IM (prime): Antibody formation level when VLP was firstly inoculated via intramuscular injection

VLP IM (boast): Antibody formation level when re-inoculated with VLP by intramuscular injection

VLP IN (prime): Antibody formation level when VLP was first inoculated through respiratory tract

VLP IN (prime): Antibody formation level when VLP is re-inoculated through respiratory tract

NAIVE: negative control (mouse serum not used for anything)

* PRIME: Serum sample after 3 weeks after the first inoculation

* BOAST: Serum sample after 3 weeks after re-vaccination under the same conditions as PRIME

FIG. 2 shows the immunological response of the vaccine through intramuscular injection and respiratory injection of VLP.

Test Example 3: Confirmation of VLP by electron microscope

In order to observe the formation of virus - like particles, negative staining was performed and observed with an electron microscope. 10 μl of VLP was adsorbed on a Formvar-carbon coated EM grid for 1 min. After negative staining for 15 sec with 2% Uranyl acetate, the grid was dried for 40 min. And then examined using transmission electron microscopy (JEM-1011, JEOL, Japan) at a magnification of 200,000 times. The above results are shown in FIG.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be possible. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description and all changes or modifications derived from the meaning and scope of the claims and their equivalents are to be construed as being included within the scope of the present invention do.

<110> SUNGSHIN WOMEN'S UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> VIRUS LIKE PARTICLE VACCINE FOR H5 INFLUENZA VIRUS AND THE METHOD          FOR THE SAME <130> P20160041KR <160> 6 <170> KoPatentin 3.0 <210> 1 <211> 1704 <212> DNA <213> Influenzae virus <400> 1 atggagaaaa tagtgcttct tcttgcagtg gttagccttg ttaaaagtga tcagatttgc 60 attggttacc atgcaaacaa ctcaacaaag caggttgaca cgataatgga aaaaaacgtc 120 actgttacac atgcccaaga catactggaa aagacacaca acgggaagct ctgcgatctt 180 aatggagtga agcccctgat tctaaaggat tgtagcgtag ctgggtggct ccttggaaat 240 ccaatgtgcg acgagttcat cagggtgccg gaatggtctt acatcgtgga gagggctaac 300 ccagccaacg acctctgtta cccagggacc ctcaatgact atgaggaatt gaaacaccta 360 ttgagcagaa taaatcattt tgagaaaact ctgatcatcc ccaagagttc ttggcccaat 420 catgaaacat cattaggggt gagcgcagca tgtccatacc agggagcatc ctcatttttc 480 agaaatgtgg tatggctcat caaaaagaac gatgcatacc cgacaataaa gataagctac 540 aataatacca atcgggaaga tcttttgata ctgtggggga ttcatcattc caacaatgca 600 gcagagcaga caaatctcta taaaaaccca gacacttatg tttccgttgg gacatcaaca 660 ttaaaccaga gattggtgcc caaaatagct actagatccc aagtaaacgg gcaacgtgga 720 agaatggatt tcttctggac aattttaaaa ccgaatgatg caatccactt tgagagtaat 780 ggaaatttca ttgctccgga atatgcctac aaaattgtca agaaagggga ctcaacaatt 840 atgaaaagtg aagtggaata tggccactgc aacaccaaat gtcaaactcc aataggggcg 900 ataaactcta gcatgccatt ccacaatata caccctctca ccatcgggga atgccccaaa 960 tacgtgaagt caaacaaatt agtccttgcg actgggctca gaaatagtcc tctaagagaa 1020 agaagaagaa aaagaggact atttggagct atagcagggt ttatagaggg aggatggcag 1080 ggaatggtag acggttggtt tgggtgcccc catagcaatg agcaggggag tgggtacgct 1140 gcagacaaag aatccaccca aaaggcaata gatggagtta ccaataaggt caactcaatc 1200 attgacaaaa tgaacactca atttgaggcc gttggaaggg aatttaataa cttagaaagg 1260 agaatagaga atttaaacaa gaaaatggaa gacggattcc tagatgtctg gacttataat 1320 gctgaacttt tagttctcat ggaaaatgag agaactctag atttccatga ctcaaatgtc 1380 aagaaccttt acgacaaagt ccgactacag cttagggata atgcaaagga gctgggtaat 1440 ggttgtttcg agttctatca caaatgtgat aaagaatgta tggaaagcgt aagaaatggg 1500 acgtatgact accctaagta ttcagaagaa gcaagattaa aaagagaaga aataagcgga 1560 gtgaaattag aatcaatagg aacttaccaa atactgtcaa tttattcaac agtggcgagt 1620 tccctagcac tggcaatcat agtggctggt ctatctttat ggatgtgctc taatgggtcg 1680 ctacaatgca gaatttgcat ctaa 1704 <210> 2 <211> 567 <212> PRT <213> Influenzae virus <400> 2 Met Glu Lys Ile Val Leu Leu Leu Ala Val Val Ser Leu Val Lys Ser   1 5 10 15 Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Lys Gln Val              20 25 30 Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile          35 40 45 Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asn Gly Val Lys      50 55 60 Pro Leu Ile Leu Lys Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn  65 70 75 80 Pro Met Cys Asp Glu Phe Ile Arg Val Pro Glu Trp Ser Tyr Ile Val                  85 90 95 Glu Arg Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Thr Leu Asn             100 105 110 Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn His Phe Glu         115 120 125 Lys Thr Leu Ile Ile Pro Lys Ser Ser Trp Pro Asn His Glu Thr Ser     130 135 140 Leu Gly Val Ser Ala Ala Cys Pro Tyr Gln Gly Ala Ser Ser Phe Phe 145 150 155 160 Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asp Ala Tyr Pro Thr Ile                 165 170 175 Lys Ile Ser Tyr Asn Asn Thr Asn Arg Glu Asp Leu Leu Ile Leu Trp             180 185 190 Gly Ile His His Ser Asn Asn Ala Ala Glu Gln Thr Asn Leu Tyr Lys         195 200 205 Asn Pro Asp Thr Tyr Val Ser Val Gly Thr Ser Thr Leu Asn Gln Arg     210 215 220 Leu Val Pro Lys Ile Ala Thr Arg Ser Gln Val Asn Gly Gln Arg Gly 225 230 235 240 Arg Met Asp Phe Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile His                 245 250 255 Phe Glu Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile             260 265 270 Val Lys Lys Gly Asp Ser Thr Ile Met Lys Ser Glu Val Glu Tyr Gly         275 280 285 His Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser     290 295 300 Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys 305 310 315 320 Tyr Val Lys Ser Asn Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Ser                 325 330 335 Pro Leu Arg Glu Arg Arg Arg Lys Arg Gly Leu Phe Gly Ala Ile Ala             340 345 350 Gly Phe Ile Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Phe Gly         355 360 365 Cys Pro His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu     370 375 380 Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser Ile 385 390 395 400 Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe Asn                 405 410 415 Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met Glu Asp Gly             420 425 430 Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu         435 440 445 Asn Glu Arg Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr     450 455 460 Asp Lys Val Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn 465 470 475 480 Gly Cys Phe Glu Phe Tyr His Lys Cys Asp Lys Glu Cys Met Glu Ser                 485 490 495 Val Arg Asn Gly Thr Tyr Asp Tyr Pro Lys Tyr Ser Glu Glu Ala Arg             500 505 510 Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly Thr         515 520 525 Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala Leu     530 535 540 Ala Ile Val Ala Gly Leu Ser Leu Trp Met Cys Ser Asn Gly Ser 545 550 555 560 Leu Gln Cys Arg Ile Cys Ile                 565 <210> 3 <211> 1413 <212> DNA <213> Influenzae virus <400> 3 atgaatccaa atcagaaaat agtaaccatt ggctccattt cattagggtt ggttgtattc 60 aatgttcttc tgcatgccgt gagcatcata ttaacagtgt tagccctggg gaagagtgaa 120 aacaatggaa tctgcaatgg aactgtagtg agggaataca atgaaacagt tagaatagag 180 aaagtgactc aatggtacaa tactagcgta gtcgaatatg taccgcattg gaatgagggc 240 acttatataa ataacaccga accaatatgt gatgtcaagg gctttgcacc tttttccaag 300 gacaacggaa taagagttgg ctccagggga catatttttg tcataagaga gcctttcgtc 360 tcttgttcac ctgtagagtg caggactttc ttcctcactc aaggatctct actcaatgac 420 aaacactcaa atggaacagt gaaggataga agcccattca gaactctcat gagtgtcgaa 480 gtgggccaat cacccaatgt atatcaagcc aggtttgaag ctgtggcatg gtcagcaaca 540 gcctgtcatg atggtaagaa gtggatggcg attggtgtaa cagggccaga ttctaaagca 600 gtagcagtag ttcattacgg aggggtgcct actgacgttg ttaactcctg ggcaggagat 660 atattaagaa ctcaggagtc atcttgtact tgcattcaag gtaattgtta ttgggtaatg 720 actgacggtc ctgccaatag acaggcgcag tatagaatat acaaagcaaa tcaaggcaaa 780 ataattggcc gaacagatgt tagctttagt ggaggacata ttgaggaatg ttcttgttat 840 ccaaatgatg gtaaagtgga atgcgtgtgt agagacaact ggacgggaac taacaggcct 900 gtgctaatta tttcgcctga tctctcttac agggttgggt atttatgtgc agggttgccc 960 agtgacactc caagagggga agatactcaa tttgtcggtt catgcactag tcccatggga 1020 aatcagggat atggcgtaaa agggttcggg tttcgacagg gaactgatgt gtgggtgggg 1080 cggacaatta gtcgaacctc cagatcagga tttgaaataa taaggataaa gaatggttgg 1140 acgcaaacaa gcaaagaaca gattagaaga caggtggttg ttgataactc gaattggtcg 1200 ggatacagtg ggtctttcac tttaccagta gaattgtctg ggagggaatg tttggttccc 1260 tgtttttggg tcgaaatgat cagaggtagg ccagaagaga gaacaatctg gacctctagt 1320 agctccattg taatgtgtgg agttgattat gaaattgccg attggtcatg gcacgatgga 1380 gctattcttc cctttgacat cgataagatg taa 1413 <210> 4 <211> 470 <212> PRT <213> Influenzae virus <400> 4 Met Asn Pro Asn Gln Lys Ile Val Thr Ile Gly Ser Ile Ser Leu Gly   1 5 10 15 Leu Val Val Phe Asn Val Leu Leu His Ala Val Ser Ile Ile Leu Thr              20 25 30 Val Leu Ala Leu Gly Lys Ser Glu Asn Asn Gly Ile Cys Asn Gly Thr          35 40 45 Val Val Arg Glu Tyr Asn Glu Thr Val Arg Ile Glu Lys Val Thr Gln      50 55 60 Trp Tyr Asn Thr Ser Val Glu Tyr Val Pro His Trp Asn Glu Gly  65 70 75 80 Thr Tyr Ile Asn Asn Thr Glu Pro Ile Cys Asp Val Lys Gly Phe Ala                  85 90 95 Pro Phe Ser Lys Asp Asn Gly Ile Arg Val Gly Ser Arg Gly His Ile             100 105 110 Phe Val Ile Arg Glu Pro Phe Val Ser Cys Ser Pro Val Glu Cys Arg         115 120 125 Thr Phe Leu Thr Gln Gly Ser Leu Leu Asn Asp Lys His Ser Asn     130 135 140 Gly Thr Val Lys Asp Arg Ser Pro Phe Arg Thr Leu Met Ser Val Glu 145 150 155 160 Val Gly Gln Ser Pro Asn Val Tyr Gln Ala Arg Phe Glu Ala Val Ala                 165 170 175 Trp Ser Ala Thr Ala Cys His Asp Gly Lys Lys Trp Met Ala Ile Gly             180 185 190 Val Thr Gly Pro Asp Ser Lys Ala Val Ala Val Val His Tyr Gly Gly         195 200 205 Val Pro Thr Asp Val Val Asn Ser Trp Ala Gly Asp Ile Leu Arg Thr     210 215 220 Gln Glu Ser Ser Cys Thr Cys Ile Gln Gly Asn Cys Tyr Trp Val Met 225 230 235 240 Thr Asp Gly Pro Ala Asn Arg Gln Ala Gln Tyr Arg Ile Tyr Lys Ala                 245 250 255 Asn Gln Gly Lys Ile Ile Gly Arg Thr Asp Val Ser Phe Ser Gly Gly             260 265 270 His Ile Glu Glu Cys Ser Cys Tyr Pro Asn Asp Gly Lys Val Glu Cys         275 280 285 Val Cys Arg Asp Asn Trp Thr Gly Thr Asn Arg Pro Val Leu Ile Ile     290 295 300 Ser Pro Asp Leu Ser Tyr Arg Val Gly Tyr Leu Cys Ala Gly Leu Pro 305 310 315 320 Ser Asp Thr Pro Arg Gly Glu Asp Thr Gln Phe Val Gly Ser Cys Thr                 325 330 335 Ser Pro Met Gly Asn Gln Gly Tyr Gly Val Lys Gly Phe Gly Phe Arg             340 345 350 Gln Gly Thr Asp Val Trp Val Gly Arg Thr Ile Ser Arg Thr Ser Arg         355 360 365 Ser Gly Phe Glu Ile Ile Arg Ile Lys Asn Gly Trp Thr Gln Thr Ser     370 375 380 Lys Glu Gln Ile Arg Arg Gln Val Val Val Asp Asn Ser Asn Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Thr Leu Pro Val Glu Leu Ser Gly Arg Glu                 405 410 415 Cys Leu Val Pro Cys Phe Trp Val Glu Met Ile Arg Gly Arg Pro Glu             420 425 430 Glu Arg Thr Ile Trp Thr Ser Ser Ser Ser Val Met Cys Gly Val         435 440 445 Asp Tyr Glu Ile Ala Asp Trp Ser Trp His Asp Gly Ala Ile Leu Pro     450 455 460 Phe Asp Ile Asp Lys Met 465 470 <210> 5 <211> 759 <212> DNA <213> Influenzae virus <400> 5 atgagtcttc taaccgaggt cgaaacgtac gtactctcta tcatcccgtc aggccccctc 60 aaagccgaga tcgcacagag acttgaagat gtctttgcag ggaagaacac cgatcttgag 120 gttctcatgg aatggctaaa gacaagacca atcctgtcac ctctgactaa ggggatttta 180 ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc ttaatgggaa cggggatcca aataacatgg acaaagcagt taaactgtat 300 aggaagctca agagggagat aacattccat ggggccaaag aaatctcact cagttattct 360 gctggtgcac ttgccagttg tatgggcctc atatacaaca ggatgggggc tgtgaccact 420 gaagtggcat ttggcctggt atgtgcaacc tgtgaacaga ttgctgactc ccagcatcgg 480 tctcataggc aaatggtgac aacaaccaat ccactaatca gacatgagaa cagaatggtt 540 ttagccagca ctacagctaa ggctatggag caaatggctg gatcgagtga gcaagcagca 600 gaggccatgg aggttgctag tcaggctaga caaatggtgc aagcgatgag aaccattggg 660 actcatccta gctccagtgc tggtctgaaa aatgatcttc ttgaaaattt gcaggcctat 720 cagaaacgaa tgggggtgca gatgcaacgg ttcaagtga 759 <210> 6 <211> 252 <212> PRT <213> Influenzae virus <400> 6 Met Ser Leu Leu Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro   1 5 10 15 Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe              20 25 30 Ala Gly Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr          35 40 45 Arg Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe      50 55 60 Thr Leu Thr Val Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val  65 70 75 80 Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala                  85 90 95 Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala             100 105 110 Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met         115 120 125 Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val Ala Phe     130 135 140 Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp Ser Gln His Arg 145 150 155 160 Ser His Arg Gln Met Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu                 165 170 175 Asn Arg Met Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met             180 185 190 Ala Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln         195 200 205 Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Ser Ser     210 215 220 Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr 225 230 235 240 Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys                 245 250

Claims (9)

A hemagglutinin (HA) protein and a neuraminidase (NA) protein derived from the H5N8 virus; And
An M1 protein derived from the H1N1 virus;
(VLP). &Lt; / RTI &gt;
The method according to claim 1,
Wherein the H5N8 virus is A / mallard / Korea / W452 / 2014 (H5N8).
The method according to claim 1,
Wherein the H1N1 virus is A / Puerto Rico / 8-V24 / 1934 (H1N1).
The method according to claim 1,
Wherein said hemagglutinin (HA) protein is defined by the amino acid sequence of SEQ ID NO: 2.
The method according to claim 1,
Wherein said neuraminidase (NA) protein is defined by the amino acid sequence of SEQ ID NO: 4.
The method according to claim 1,
Wherein said M1 protein is defined by the amino acid sequence of SEQ ID NO: 6.
A vaccine composition comprising the virus-like particle according to claim 1 as an active ingredient.
8. A method for preventing avian influenza comprising administering to a bird a vaccine composition according to claim 7.
Producing a recombinant vector comprising a gene of a hemagglutinin (HA) protein derived from the H5N8 virus;
Transposing the recombinant plasmid to a Bacmid expressing a neuraminidase (NA) protein derived from the H5N8 virus;
Transfecting said recombinant Bacmid into insect cells;
Obtaining a recombinant baculovirus from the insect cell;
Insecting insect cells with baculovirus expressing the M1 protein derived from the recombinant baculovirus and the H1N1 virus; And
Separating the hemagglutinin (HA) protein, the neuraminidase (NA) protein, and the virus-like particle that forms the envelope of the M1 protein in the insect cell;
Like particles.

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