KR101729387B1 - Method for manufacturing influenza vaccine material M1-M2-NA-HA virus-like-particle using insect cell expression system - Google Patents

Abstract

The present invention relates to a method for producing an influenza virus-like particle vaccine material using an insect cell expression system. By using the present invention, M1-M2-NA-HA virus-like particles of influenza virus, which is a material that can be used for an influenza vaccine, can be stably and efficiently produced.

Description

[0001] The present invention relates to a method for producing influenza vaccine M1-M2-NA-HA virus-like particle using an insect cell expression system,

The present invention relates to a method for producing influenza vaccine M1-M2-NA-HA virus-like particles using an insect cell expression system.

Influenza (influenza) is a respiratory disease that spreads through the respiratory system of people and animals (birds, pigs, dogs, horses, etc.) by the influenza virus. Human influenza occurs every year in the 10-20% of the world, and is highly contagious and tends to spread worldwide on an annual basis. Symptoms of influenza include high fever, headache, muscle aches, inflammation of the throat, pain, cough and other respiratory diseases. Severe cases can lead to deaths of elderly people and chronic disease holders.

The influenza vaccine for humans is mainly a vaccine containing HA protein isolated from influenza virus. However, since only one antigen is used, immunogenicity is somewhat low. On the other hand, attenuated vaccines have high immunogenicity but low safety due to the risk of infection. Therefore, how to increase immunogenicity and safety at the same time is an important task in influenza vaccine production.

In the case of human influenza vaccines, vaccines should be prepared by predicting and anticipating strains that are expected to become popular in the year. Therefore, it is not only necessary to produce vaccine repeatedly in accordance with the strain, but also the problem that the existing vaccine is not effective when the actual strain is inconsistent with the strain used for the vaccine production, I have. Therefore, in order to overcome this problem, a lot of studies are being conducted to develop an influenza vaccine by selecting an antigen having a wide range of defense ability in humans and animals.

Currently, the production of influenza vaccine is manufactured using fertilized eggs. This vaccine manufacturing method takes a relatively long time (more than 6 months) and its production process is complicated, so as a vaccine manufacturing method for quickly responding to the spread of influenza pandemic It is not appropriate. In addition, there is a disadvantage that the vaccine may contain egg protein and cause local or systemic allergy. Thus, there is a need to develop a rapid and flexible vaccine manufacturing method.

Korean Patent Publication No. 10-2009-0029851 (title of the invention: influenza vaccine) Korean Patent No. 10-1316350 (Title of the Invention: Method for producing influenza vaccine composition)

The present invention relates to a recombinant plasmid vector capable of expressing an influenza M1-M2-NA-HA virus-like particle, an insect cell transformed with the recombinant vector, an influenza M1-M2-NA-HA A method for producing virus-like particles, and an influenza virus vaccine composition comprising the virus-like particles.

The present invention provides a recombinant plasmid vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2.

The present invention also provides a recombinant plasmid vector comprising the influenza NA gene of SEQ ID NO: 3 and the influenza HA gene of SEQ ID NO:

The M1, M2, NA, HA genes may be obtained from the influenza H1N1 virus and may be obtained, for example, from the influenza H1N1 virus [Influenza A / Korea / 01/2009 (H1N1)]. However, since the genes M1, M2, NA, and HA correspond to genes that exist regardless of the serotype of influenza, the source of the gene is not limited thereto.

The recombinant vector of the present invention may be a plasmid vector. However, the vectors that can be used in the present invention are not limited thereto, and any vector that can be used for expression in insect cells can be used.

The plasmid vector of the present invention may further comprise the promoter gene of SEQ ID NO: 5 and the terminator gene of SEQ ID NO: However, the types of the promoter gene and terminator gene that can be used in the recombinant vector of the present invention are not limited thereto.

The present invention also provides a recombinant plasmid vector comprising the influenza M1 gene of SEQ ID NO: 1, the influenza M2 gene of SEQ ID NO: 2, the influenza NA gene of SEQ ID NO: 3 and the influenza HA gene of SEQ ID NO: Thereby providing transformed insect cells.

The insect cell may be a cell of Trichoplusia ni, preferably a BT1-Tn-5B1-4 cell. However, the type of insect cells is not limited thereto. As one embodiment of the present invention, insect cells corresponding to Lepidopteran insect cells are all available, for example, LD652Y cells of Lymantria dispar, Sf9 cells of Spodoptera frugiperda Insect cells selected from the group consisting of Sf21 cells of Spodoptera frugiperda, Kc1 cells of Drosophila, SL2 cells of Drosophila, and mosquito cell lines. Can be used.

(A) preparing a recombinant plasmid vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2;

(B) producing a recombinant plasmid vector comprising the influenza NA gene of SEQ ID NO: 3 and the influenza HA gene of SEQ ID NO: 4;

Transforming an insect cell with the recombinant plasmid vector of (a) and the recombinant plasmid vector of (b) above; And

M2-NA-HA virus-like particle (VLP) from the culture medium in which the transformed insect cells have been cultivated in order to obtain influenza M1-M2-NA-HA virus-like particles Thereby producing a virus-like particle.

In the present invention, the cell culture medium generally means that cells are removed from the culture medium in which the cells are cultured.

Matrix protein 1 (M1), matrix protein 2 (M2), neuraminidase (NA) and hemagglutinin (HA) are the structural proteins of influenza virus that can be used to develop influenza subunit vaccines. The M1 protein is a matrix protein that forms a coat inside the envelope of influenza virus and the M2 protein is a substrate protein that binds to the M1 protein coat to form an ion channel. Both NA and HA are representative antigens of influenza present on the virus surface. In the transformed insect cells of the present invention, M1 protein, M2 protein, NA protein and HA protein are expressed and secreted into the culture medium as M1-M2-NA-HA virus-like particles. Virus-like particles contain one or more viral structural proteins, typically containing envelope proteins and matrix proteins, but not genetic material of viruses. Thus, the desired immune response to the virus can be derived from the body without the risk of infection.

The present invention provides an influenza vaccine composition comprising influenza M1-M2-NA-HA virus-like particles.

Virus-like particles act as antigens in the body and react with antigen-labeled cells, such as dendritic cells, to label the T or B immune cells with the antigen. Virus-like particles have a structure similar to the original virus but do not have a genetic material, so that it is impossible to multiply and thus safety can be expected for use in applications such as vaccines. Viral surface proteins inserted on the surface of virus-like particles have higher antigenicity than purely isolated recombinant proteins and can form effective neutralizing antibodies. A representative example of a vaccine using virus-like particles is the human papilloma virus type (HPV-16) L1 vaccine produced by GlaxoSmithKline (GSK).

These virus-like particle-based vaccines are known to have immune efficacy like living, replicable viruses. In other words, virus-like particles have the ability to effectively activate immune cells, leading to induction of high antibody formation and effective protective immunity (Song JM, et al., Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like Virology 405: 165-175, (2010); Sailaja G et al., Human immunodeficiency virus-like particles activate multiple types of immune cells. Virology 362: 331-341, (2007)). That is, different immunogenicity can be reinforced by inserting and substituting mutant proteins or homologous proteins of the viral proteins into existing genes. In addition, cross protection against influenza is improved. The M1-M2-NA-HA virus-like particles of the present invention can be used as a material for producing a tetravalent influenza vaccine.

In addition, using the recombinant plasmid vector of the present invention and insect cells transformed with the vector, the production process is not complicated as compared with the conventional influenza virus, animal cell, and baculovirus vector production method, Lt; RTI ID = 0.0 > influenza < / RTI > vaccine. Stably transformed insect cells can produce proteins with post-translational modifications (glycosylation, phosphorylation, addition of fatty acids) similar to those expressed in animal cells, and also have high protein expression and simultaneous expression of multiple proteins There is an advantage that it can be discharged out of the cell.

The vaccine composition of the present invention may be administered prior to influenza seasoning every year and may be administered by subcutaneous or intramuscular injection of 0.1 ml to 1.0 ml. However, the frequency of administration, the timing of administration, the dosage and route of administration are illustrative and not restrictive.

The present invention can be used to stably and efficiently produce M1-M2-NA-HA virus-like particles of influenza virus, which is a material that can be used for an influenza vaccine.

FIG. 1 is a schematic diagram of insertion of a DNA fragment of Orgyia pseudotsugata immediate-early 2 promoter (pOpIE2) and Orgyia pseudotsugata immediate-early 2 poly A terminator (OpIE2 pA) into pIZT / V5-His vector.
2 is a schematic diagram of a pIZT2 / M1-M2 vector constructed by inserting influenza virus M1 and M2 protein genes into the recombination vector of FIG.
FIG. 3 is a schematic diagram of inserting pOpIE2 and OpIE2 pA DNA fragments into a pCoHygro vector.
Fig. 4 is a schematic diagram of a vector constructed by inserting influenza virus NA and HA protein gene into the recombination vector of Fig. 3; Fig.
FIG. 5 shows the results of confirming M1, M2, NA and HA proteins in extracts of BT1-Tn-5B1-4 / M1-M2-NA-HA cells.
FIG. 6 shows the results of confirming M1, M2, NA and HA proteins in the culture medium of BT1-Tn-5B1-4 / M1-M2-NA-HA cells.
FIG. 7 shows the results of Western blotting in the culture medium of BT1-Tn-5B1-4 / M1-M2-NA-HA cells to confirm that most of the M1, M2, NA and HA proteins are present in fractions 4 and 5.
FIG. 8 shows the results of confirming the presence of virus-like particles by observing the precipitates of fractions 4 and 5 with an electron microscope.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1 Preparation of Recombinant Plasmid Vector Simultaneously Expressing M1 and M2 Protein and Transformation of Insect Cells

1-1. Recombinant plasmid vector expression simultaneously expressing M1 and M2 proteins

Promoters and terminators that regulate the expression of each gene are required in order to simultaneously express the M1 and M2 proteins. Since there is only one promoter and one terminator each capable of regulating the expression of the recombinant protein in the pIZT / V5-His vector (Invitrogen, USA), the promoter and terminator present in the pIZT / V5- And cloned into pIZT / V5-His vector, respectively.

(Taq polymerase (Takara, Japan) and the following Table 1 were used as the templates and the template for the Orgyia pseudotsugata immediate-early 2 promoter (pOpIE2) and the Orgyia pseudotsugata immediate-early 2 poly A terminator (OpIE2 pA) contained in the pIZT / V5- PCR was performed to amplify the pOpIE2 DNA fragment and the OpIE2 pA DNA fragment.

[Table 1]

The pOpIE2 DNA fragment and the OpIE2 pA DNA fragment were cloned into the EcoR I / EcoR V and EcoR V / Not I restriction sites of the pIZT / V5-His vector, an insect cell expression vector, respectively, to regulate the expression of the recombinant protein A recombinant pIZT2 vector with a pair of promoter-terminators was constructed (see also the schematic diagram of Fig. 1).

(SEQ ID NO: 1) and M2 (SEQ ID NO: 2) protein fragments were obtained through RT-PCR using the influenza H1N1 virus [Influenza A / Korea / 01/2009 (H1N1)] genome as a template. The M1 and M2 protein DNA fragments were respectively cloned into the Spe I / EcoR I and Not I / Xba I restriction sites of the pIZT2 vector constructed above to construct a recombinant pIZT2 / M1-M2 vector (see the schematic diagram of FIG. 2 ).

1-2. Transformation of insect cells

The recombinant pIZT2 / M1-M2 vector (2.2 μg) was mixed with 250 μl of Express Five Serum Free Medium (Invitrogen) and 250 μl of Express Five SFM mixed with 18 μl of Cellfectin II reagent (Invitrogen) The mixture was allowed to react at room temperature for 10 minutes. The pIZT2 / M1-M2 vector / Cellfectin II reagent mixture was added to a well of a 6 well plate in which BT1-Tn-5B1-4 cells (Invitrogen) of 1.8 x 10 ⁶ Trichoplusia ni were dispensed per well Transfection was induced by culturing for 2 days.

Since the zeocin-GFF sequence is present in the pIZT / V5-His vector, cells transfected with the pIZT2 / M1-M2 vector are resistant to zeocin antibiotics. Therefore, the cultured insect cells were collected and added to Express Five SFM medium supplemented with 300 μg / ml of zeocin (Invitrogen) antibiotic, and the insect cells resistant to zeocin antibiotics were cultured for 5 weeks Screening period.

Example 2 Preparation and Transformation of a Recombinant Plasmid Vector Simultaneously Expressing NA and HA Protein

2-1. Production of recombinant plasmid vectors expressing NA and HA proteins simultaneously

Using the LA Taq polymerase (Takara, Japan) and the primers shown in Table 1 above, using the Orgyia pseudotsugata immediate-early 2 promoter (pOpIE2) and Orgyia pseudotsugata immediate-early 2 poly A terminator (OpIE2 pA) contained in the pCoHygro vector as a template PCR was performed to amplify the pOpIE2 DNA fragment and the OpIE2 pA DNA fragment.

The pOpIE2 DNA fragment and the OpIE2 pA DNA fragment were cloned into the SphI / EcoRV and EcoRV / SalI restriction sites of the pCoHygro vector for insect cell expression, respectively, to give recombinant proteins with two pairs of promoter-terminator pICHTx2 vector was constructed (see also the schematic diagram of Fig. 3).

DNA fragments of NA (SEQ ID NO: 3) and HA (SEQ ID NO: 4) were respectively obtained by RT-PCR using the influenza H1N1 virus [Influenza A / Korea / 01/2009 (H1N1)] genome as templates, and pICHTx2 The vector was cloned into the SpeI / EcoRI and NotI / SacII restriction sites, respectively, to construct a recombinant pICHTx2 / NA-HA vector (see the schematic diagram of FIG. 4).

2-2. Transformation of insect cells

BT1-Tn-5B1-4 cells simultaneously expressing the M1 and M2 proteins prepared in Example 1 were dispensed into wells of a 6-well plate. The recombinant pICHTx2 / NA-HA vector (2.2 μg) prepared in 2-1 above was mixed with 250 μl of Express Five Serum Free Medium (Invitrogen) and 250 μl of Cellfectin II reagent (Invitrogen) Of Express Five SFM and reacted at room temperature for 10 minutes. The recombinant pICHTx2 / NA-HA vector mixture was added to the fractionated BT1-Tn-5B1-4 cells and cultured for 2 days to induce transformation.

Since hygromycin resistance selectable markers are present in the pICHTx2 / NA-HA vector, the transformed cells are resistant to hygromycin antibiotics. Thus, the cultured insect cells were recovered and added to Express Five SFM medium supplemented with 300 μg / ml of hygromycin B (Invitrogen) antibiotic, insect cells having resistance to hygromycin B Were selected through a screening period of about 5 weeks. The selected recombinant insect cells were named BT1-Tn-5B1-4 / M1-M2-NA-HA cells.

<Example 3> Expression of M1, M2, HA and NA proteins in insect cell extract

M2, HA, and NA proteins were expressed in BT1-Tn-5B1-4 / M1-M2-NA-HA cells.

BT1-Tn-5B1-4 / M1-M2-NA-HA cells were added to a 6-well plate at a concentration of 1 × 10 ⁶ cells / well and cultured in a 27 ° C. incubator for 4 days. Cells were recovered from the cell culture and proteins were extracted from the cells. Protein extracts were analyzed by Western blotting with anti-H1N1 M1 antibody, anti-H1N1 M2 antibody, anti-H1N1 NA antibody and anti-H1N1 HA antibody, and the recombinant HA, NA, M1 and M2 proteins were stably expressed (See Fig. 5). The sizes of the expressed M1, M2, HA and NA proteins were 28, 17, 60 and 70 kDa, respectively.

M2, HA, and NA proteins of H1N1 virus were stably expressed in BT1-Tn-5B1-4 / M1-M2-NA-HA cells.

Example 4 Production of M1-M2-NA-HA Virus-Like Particles in Insect Cell Culture Medium

4-1. Western blot

5 × 10 ⁶ BT1-Tn-5B1-4 / M1-M2-NA-HA cells were added to a T-25 culture flask (SPL, Korea) and cultured in a 27 ° C. incubator for 4 days. The cell culture medium was centrifuged to obtain a cell culture medium from which the cells had been removed, and the obtained cell culture medium was added to a centrifuge vessel having a sucrose cushion of 30% (w / w), followed by centrifugation at 32,000 rpm for 2 hours . Western blotting was performed using anti-H1N1 M1, anti-H1N1 M2, anti-H1N1 NA and anti-H1N1 HA antibodies as a precipitate obtained by centrifugation. As shown in FIG. 6, it was found that M1, M2, NA and HA proteins were present in 30% sucrose cushion precipitate. When proteins M1, M2, NA, and HA do not constitute virus-like particles, they are secreted out of the cell and can not be present in the cell culture medium. From these results, it can be seen that the proteins are produced in transformed insect cells, I could.

The 30% sucrose cushion precipitate was added to a centrifuge vessel having a sucrose density gradient of 30-60% and fractionated by centrifugation at 32,000 rpm for 3 hours. Western blotting was performed to determine the presence of recombinant M1, M2, (Fig. 7). As a result, it was found that most of M1, M2, NA and HA proteins were present together (fractions 4 and 5). From these results, it was found that the proteins constituted M1-M2-NA-HA virus-like particles.

4-2. TEM observation

Fractions 4 and 5 obtained in 4-1 were attached to a nickel grid for 1 min, stained with 2% uranyl acetate for 1 min, observed with an energy filtration transmission electron microscope (EF-TEM) (Fig. 8).

From the above results, M1-M2-NA-HA virus-like particles were produced in the transformed insect cells and secreted into the culture medium.

Example 5 Production of M1-M2-NA-HA Virus-like Particles

100 ml of culture medium containing BT1-Tn-5B1-4 / M1-M2-NA-HA cells was added to a 30% (w / v) sucrose cushion centrifuge and centrifuged at 32,000 rpm for 2 hours Respectively.

The amount of protein present in the precipitate was determined. In addition, since it was confirmed that M1-M2-NA-HA exists as virus-like particles in Example 3, the activity of M1-M2-NA-HA virus-like particles was indirectly confirmed by confirming HA activity present in the precipitate .

The results are shown in Table 2 below.

[Table 2]

As shown in Table 2, a total of 0.07 mg of protein was present per 100 ml of the culture medium, and HA activity was measured to be very high, 100 HAU / mg.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Method for manufacturing influenza vaccine material M1-M2-NA-HA          virus-like-particle using insect cell expression system <130> P15-057-KHU <160> 6 <170> Kopatentin 2.0 <210> 1 <211> 759 <212> DNA <213> Artificial Sequence <220> <223> Influenza M1 gene <400> 1 atgagtcttc taaccgaggt cgaaacgtac gttctttcta tcatcccgtc aggccccctc 60 aaagccgaga tcgcgcagag actggaaagt gtctttgcag gaaagaacac agatcttgag 120 gctctcatgg aatggctaaa gacaagacca atcttgtcac ctctgactaa gggaatttta 180 ggatttgtgt tcacgctcac cgtgcccagt gagcgaggac tgcagcgtag acgctttgtc 240 caaaatgccc taaatgggaa tggggacccg aacaacatgg atagagcagt taaactatac 300 aagaagctca aaagagaaat aacgttccat ggggccaagg aggtgtcact aagctattca 360 actggtgcac ttgccagttg catgggcctc atatacaaca ggatgggaac agtgaccaca 420 gaagctgctt ttggtctagt gtgtgccact tgtgaacaga ttgctgattc acagcatcgg 480 tctcacagac agatggctac taccaccaat ccactaatca ggcatgaaaa cagaatggtg 540 ctggctagca ctacggcaaa ggctatggaa cagatggctg gatcgagtga acaggcagcg 600 gaggccatgg aggttgctaa tcagactagg cagatggtac atgcaatgag aactattggg 660 actcatccta gctccagtgc tggtctgaaa gatgaccttc ttgaaaattt gcaggcctac 720 cagaagcgaa tgggagtgca gatgcagcga ttcaagtga 759 <210> 2 <211> 294 <212> DNA <213> Artificial Sequence <220> <223> Influenza M2 gene <400> 2 atgagtcttc taaccgaggt cgaaacgcct accagaagcg aatgggagtg cagatgcagc 60 gattcaagtg atcctctcgt cattgcagca aatatcattg ggatcttgca cctgatattg 120 tggattactg atcgtctttt tttcaaatgt atttatcgtc gctttaaata cggtttgaaa 180 agagggcctt ctacggaagg agtgcctgag tccatgaggg aagaatatca acaggaacag 240 cagagtgctg tggatgttga cgatggtcat tttgtcaaca tagagctaga gtaa 294 <210> 3 <211> 1410 <212> DNA <213> Artificial Sequence <220> <223> Influenza NA gene <400> 3 atgaatccaa accaaaagat aataaccatt ggttcggtct gtatgacaat tggaatggct 60 aacttaatat tacaaattgg aaacataatc tcaatatgga ttagccactc aattcaactt 120 gggaatcaaa atcagattga aacatgcaat caaagcgtca ttacttatga aaacaacact 180 tgggtaaatc agacatatgt taacatcagc aacaccaact ttgctgctgg acagtcagtg 240 gtttccgtga aattagcggg caattcctct ctctgccctg ttagtggatg ggctatatac 300 agtaaagaca acagtataag aatcggttcc aagggggatg tgtttgtcat aagggaacca 360 ttcatatcat gctccccctt ggaatgcaga accttcttct tgactcaagg ggccttgcta 420 aatgacaaac attccaatgg aaccattaaa gacaggagcc catatcgaac cctaatgagc 480 tgtcctattg gtgaagttcc ctctccatac aactcaagat ttgagtcagt cgcttggtca 540 gcaagtgctt gtcatgatgg catcaattgg ctaacaattg gaatttctgg cccagacaat 600 ggggcagtgg ctgtgttaaa gtacaacgga ataataacag acactatcaa gagttggaga 660 aacaatatat tgagaacaca agagtctgaa tgtgcatgtg taaatggttc ttgctttact 720 gtaatgaccg atggaccaag taatggacag gcctcataca agatcttcag aatagaaaag 780 ggaaagatag tcaaatcagt cgaaatgaat gcccctaatt atcactatga ggaatgctcc 840 tgttatcctg attctagtga aatcacatgt gtgtgcaggg ataactggca tggctcgaat 900 cgaccgtggg tgtctttcaa ccagaatctg gaatatcaga taggatacat atgccgtggg 960 attttcggag acaatccacg ccctaatgat aagacaggca gttgtggtcc agtatcgtct 1020 aatggagcaa atggagtaaa aggattttca ttcaaatacg gcaatggtgt ttggataggg 1080 agaactaaaa gcattagttc aagaaacggt tttgagatga tttgggatcc gaacggatgg 1140 actgggacag acaataactt ctcaataaag caagatatcg taggaataaa tgagtggtca 1200 ggatatagcg ggagttttgt tcagcatcca gaactaacag ggctggattg tataagacct 1260 tgcttctggg ttgaactaat cagagggcga cccaaagaga acacaatctg gactagcggg 1320 agcagcatat ccttttgtgg tgtaaacagt gacactgtgg gttggtcttg gccagacggt 1380 gctgagttgc catttaccat tgacaagtaa 1410 <210> 4 <211> 1701 <212> DNA <213> Artificial Sequence <220> <223> Influenza HA gene <400> 4 atgaaggcaa tactagtagt tctgctatat acatttgcaa ccgcaaatgc agacacatta 60 tgtataggtt atcatgcgaa caattcaaca gacactgtag acacagtact agaaaagaat 120 gtaacagtaa cacactctgt taaccttcta gaagacaagc ataacgggaa actatgcaaa 180 ctaagagggg tagccccatt gcatttgggt aaatgtaaca ttgctggctg gatcctggga 240 aatccagagt gtgaatcact ctccacagca agctcatggt cctacattgt ggaaacatct 300 agttcagaca atggaacgtg ttacccagga gatttcatcg attatgagga gctaagagag 360 caattgagct cagtgtcatc atttgaaagg tttgagatat tccccaagac aagttcatgg 420 cccaatcatg actcgaacaa aggtgtaacg gcagcatgtc ctcatgctgg agcaaaaagc 480 ttctacaaaa atttaatatg gctagttaaa aaaggaaatt catacccaaa gctcagcaaa 540 tcctacatta atgataaagg gaaagaagtc ctcgtgctat ggggcattca ccatccatct 600 actagtgctg accaacaaag tctctatcag aatgcagatg catatgtttt tgtggggtca 660 tcaagataca gcaagaagtt caagccggaa atagcaataa gacccaaagt gagggatcaa 720 gaagggagaa tgaactatta ctggacacta gtagagccgg gagacaaaat aacattcgaa 780 gcaactggaa atctagtggt accgagatat gcattcgcaa tggaaagaaa tgctggatct 840 ggtattatca tttcagatac accagtccac gattgcaata caacttgtca gacacccaag 900 ggtgctataa acaccagcct cccatttcag aatatacatc cgatcacaat tggaaaatgt 960 ccaaaatatg taaaaagcac aaaattgaga ctggccacag gattgaggaa tgtcccgtct 1020 attcaatcta gaggcctatt tggggccatt gccggtttca ttgaaggggg gtggacaggg 1080 atggtagatg gatggtacgg ttatcaccat caaaatgagc aggggtcagg atatgcagcc 1140 gacctgaaga gcacacagaa tgccattgac gagattacta acaaagtaaa ttctgttatt 1200 gaaaagatga atacacagtt cacagcagta ggtaaagagt tcaaccacct ggaaaaaaga 1260 atagagaatt taaataaaaa agttgatgat ggtttcctgg acatttggac ttacaatgcc 1320 gaactgttgg ttctattgga aaatgaaaga actttggact accacgattc aaatgtgaag 1380 aacttaatg aaaaggtaag aagccagcta aaaaacaatg ccaaggaaat tggaaacggc 1440 tgctttgaat tttaccacaa atgcgataac acgtgcatgg aaagtgtcaa aaatgggact 1500 tatgactacc caaaatactc agaggaagca aaattaaaca gagaagaaat agatggggta 1560 aagctggaat caacaaggat ttaccagatt ttggcgatct attcaactgt cgccagttca 1620 ttggtactgg tagtctccct gggggcaatc agtttctgga tgtgctctaa tgggtctcta 1680 cagtgtagga tatgtattta a 1701 <210> 5 <211> 563 <212> DNA <213> Artificial Sequence <220> <223> Orgyia pseudotsugata immediate-early 2 promoter <400> 5 tcatgatgat aaacaatgta tggtgctaat gttgcttcaa caacaattct gttgaactgt 60 gttttcatgt ttgccaacaa gcacctttat actcggtggc ctccccacca ccaacttttt 120 tgcactgcaa aaaaacacgc ttttgcacgc gggcccatac atagtacaaa ctctacgttt 180 cgtagactat tttacataaa tagtctacac cgttgtatac gctccaaata cactaccaca 240 cattgaacct ttttgcagtg caaaaaagta cgtgtcggca gtcacgtagg ccggccttat 300 cgggtcgcgt cctgtcacgt acgaatcaca ttatcggacc ggacgagtgt tgtcttatcg 360 tgacaggacg ccagcttcct gtgttgctaa ccgcagccgg acgcaactcc ttatcggaac 420 aggacgcgcc tccatatcag ccgcgcgtta tctcatgcgc gtgaccggac acgaggcgcc 480 cgtcccgctt atcgcgccta taaatacagc ccgcaacgat ctggtaaaca cagttgaaca 540 gcatctgttc gaatttaaag ctt 563 <210> 6 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> Orgyia pseudotsugata immediate-early 2 polya terminator <400> 6 gtttatctga ctaaatctta gtttgtattg tcatgtttta atacaatatg ttatgtttaa 60 atatgttttt aataaatttt ataaaataat ttcaactttt attgtaacaa cattgtccat 120 ttacacactc ctttcaagcg cgtgggatcg atgctc 156

Claims (8)

delete delete delete A recombinant plasmid vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2; And
An insect cell transformed with a recombinant plasmid vector comprising an influenza NA gene of SEQ ID NO: 3 and an influenza HA gene of SEQ ID NO:
The insect cell of claim 4, wherein the insect cell is selected from the group consisting of BT1-Tn-5B1-4 cells of Trichoplusia ni, LD652Y cells of Lymantria dispar, Spodoptera frugiperda ) From the group consisting of Sf9 cells, Spodoptera frugiperda Sf21 cells, Drosophila Kc1 cells, Drosophila SL2 cells, and mosquito cell lines Selected, insect cells.
(A) preparing a recombinant plasmid vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2;
(B) producing a recombinant plasmid vector comprising the influenza NA gene of SEQ ID NO: 3 and the influenza HA gene of SEQ ID NO: 4;
Transforming an insect cell with the recombinant plasmid vector of (a) and the recombinant plasmid vector of (b) above; And
M2-NA-HA virus-like particle (VLP) from the culture medium in which the transformed insect cells have been cultivated in order to obtain influenza M1-M2-NA-HA virus-like particles Like particles.
7. The method according to claim 6, wherein the insect cells in step (b) are selected from the group consisting of BT1-Tn-5B1-4 cells of Trichoplusia ni, LD652Y cells of Lymantria dispar, Sf9 cells of Spodoptera frugiperda, Sf21 cells of Spodoptera frugiperda, Kc1 cells of Drosophila, SL2 cells of Drosophila, and mosquito cell lines &Lt; / RTI &gt;
An influenza vaccine composition comprising influenza M1-M2-NA-HA virus-like particles produced by the method of claim 6 or 7.

Images