KR101543744B1 - Method for manufacturing influenza vaccine material M1 protein, M2 protein, and M1-M2 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, it is possible to stably and efficiently produce M1 protein, M2 protein, and M1-M2 virus-like particles of influenza virus which can be used for an influenza vaccine.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for producing influenza vaccine M1 protein, M2 protein, and M1-M2 virus-like particle using an insect cell expression system expression system}

The present invention relates to a method for producing an influenza vaccine material 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.

In the case of human influenza vaccine, vaccines should be made by anticipating and anticipating strains that are expected to be 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.

In addition, the production of influenza vaccine is currently being manufactured by using a fertilized egg (Patent No. 10-1316350, entitled " Influenza Vaccine Composition Manufacturing Method "). This vaccine manufacturing method takes a relatively long time The production process is complicated and is not suitable as a vaccine production method for quickly responding to the spread of pandemic influenza. Thus, there is a need to develop a rapid and flexible vaccine manufacturing method.

The present invention relates to a recombinant vector capable of expressing an influenza M1 and / or M2 protein, an insect cell transformed with the recombinant vector, and an insect cell transformed with the M1 protein, M2 protein, and / or M1-M2 virus Lt; RTI ID = 0.0 > a < / RTI >

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

The M1 and M2 genes may be obtained from the influenza H1N1 virus. However, since the M1 and M2 genes correspond to genes that exist regardless of the serotype of influenza, the source of the gene is not limited thereto.

The recombinant vector may further comprise a promoter gene of SEQ ID NO: 3 and a terminator gene of SEQ ID NO: 4. 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 recombinant vector may be based on the pIZT / V5-His vector, but the vectors that can be used for expression in insect cells are not limited thereto.

The present invention also provides an insect cell transformed with said recombinant vector.

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. In one embodiment of the present invention, when a recombinant vector is prepared based on a pIZT vector, insect cells corresponding to Lepidopteran insect cells are all available, preferably, LD652Y cells of Lymantria dispar, Sf9 cells of Spodoptera frugiperda, Sf21 cells of Spodoptera frugiperda, Kc1 cells of Drosophila, SL2 cells of Drosophila, and mosquitoes a mosquito cell line, and the like.

The present invention also provides a method for producing a recombinant vector comprising the steps of: (a) preparing a recombinant vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2;

(B) providing at least one selected from the group consisting of influenza M1 protein, M2 protein, and influenza M1-M2 virus-like particles, which comprises transforming insect cells with the recombinant vector.

In one embodiment of the present invention, the method may further comprise the step of obtaining influenza M1-M2 virus-like particles (VLP) from a culture medium in which the transformed insect cells are cultured, Influenza < / RTI > M1-M2 virus-like particles.

In another embodiment of the present invention, the method may further comprise the step of obtaining an influenza M1 protein and / or a M2 protein from the culture medium of the transformed insect cells, whereby influenza M1 protein and / or M2 protein Can be prepared.

The cell culture medium generally means that both the cell culture medium and the cell are included, and the cell culture medium generally means that cells are removed from the cell culture medium.

matrix protein 1 (M1) and matrix protein 2 (M2) are 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. In the transformed insect cells of the present invention, M1 protein and M2 protein are expressed, and M1-M2 virus-like particles are produced and secreted into a culture medium. Virus-like particles contain one or more viral structural proteins, typically containing envelope proteins and matrix proteins, but not genetic material of viruses.

Such virus-like particles can be prepared by methods known in the art. For example, the assembly of virus-like particles can be carried out by transforming a suitable host cell with a recombinant DNA molecule encoding the structural protein, and then culturing the recombinant DNA molecule, whereby the structural protein expressed in the cell is assembled on the cell surface and then discharged into the culture supernatant . During this process, the surface proteins contained in the virus-like particles retain their natural shape without being immobilized, so they have a virus-like shape and can elicit the desired immune response against the virus in the body.

The present invention provides an influenza vaccine comprising influenza M1-M2 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. VLP has a structure similar to that of the original virus but has no genetic material, so that it can not be propagated, so safety can be expected for use in applications such as vaccines. Viral surface proteins inserted on the surface of VLPs 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 virus-like particle of the present invention can be used as a material for producing a divalent influenza vaccine.

In addition, when the transformed insect cells of the present invention are used, the production process of the influenza vaccine using the conventional fertilized eggs is not complicated, so that an influenza vaccine capable of rapidly responding to the spread of influenza virus can be produced. 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 present invention can be used to stably and efficiently produce matrix protein 1 (M1), matrix protein 2 (M2), and M1-M2 virus-like particles of influenza virus which can be used for 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 shows Western blot analysis of recombinant M1 (A) and M2 (B) proteins in the BT1-Tn-5B1-4 cell extract of tricofluorocyanine-transformed pIZT2 / M1-M2 vector of FIG. to be.
FIG. 4 is a graph showing the effect of the pIZT2 / M1-M2 vector of FIG. 2 on the culture medium of BT1-Tn-5B1-4 cells of the tricofluorocyanine transformant obtained by centrifugation with 30% (w / w) sucrose cushion The recombinant M1 (A) and M2 (B) proteins were confirmed by Western blot.
FIG. 5 shows the results of confirming the presence of virus-like particles by observing the precipitate of FIG. 4 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> M1  And M2  Production of recombinant plasmid vector expressing protein at the same time

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.

(pOpIE2) (SEQ ID NO: 3) and Orgyia pseudotsugata immediate-early 2 poly A terminator (OpIE2 pA) (SEQ ID NO: 4) contained in the pIZT / V5- PCR was performed using the polymerase (Takara, Japan) and the primers shown in Table 1 below 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 ).

< Example  2> Transformation of insect cells

The recombinant pIZT2 / M1-M2 vector (2.2 μg) constructed in Example 1 was mixed with 250 μl Express Five SFM (Express Five Serum Free Medium, Invitrogen) and 250 μl of a mixture of 18 μl of Cellfectin II reagent (Invitrogen) Of Express Five SFM and reacted 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  3> In insect cell extract M1 , M2  Confirmation of protein expression

The following experiments were performed to confirm whether the recombinant M1 protein and the M2 protein were expressed in the transformed cells.

The transformed insect cells obtained in Example 2 were added to a 6-well plate at a rate of 1 × 10 ⁶ cells per well and cultured in a 27 ° C. incubator for 4 days. Cells were collected from the cell culture medium and proteins were extracted. The protein extracts were analyzed by Western blot using anti-H1N1 M1 antibody (AbD Serotec, UK) and anti-H1N1 M2 antibody (Gene Tex, USA) to confirm that the recombinant M1 and M2 proteins were stably expressed in the cells 3). The sizes of expressed recombinant M1 and M2 proteins were 30 and 19 kDa, respectively.

From the above results, it can be seen that the recombinant M1 and M2 proteins are stably expressed in the transformed tricofluorocyanine BT1-Tn-5B1-4 cells as described in Example 2, using the recombinant vector of Example 1.

< Example  4> insect cells On the culture medium M1 , M2  Protein and M1 - M2  Confirm production of virus-like particles

In the transformed cells, when the M1-M2 protein forms virus-like particles, it is secreted outside the cell. Therefore, the following experiment was conducted to confirm formation of virus-like particles by the M1-M2 protein.

5 x 10 ⁶ transformed insect cells prepared in Example 2 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 and anti-H1N1 M2 antibodies as the precipitate obtained by centrifugation. As shown in FIG. 4, it was found that M1 and M2 proteins were present in 30% sucrose cushion precipitate. When M1 and M2 proteins do not constitute viral-like particles, they are secreted out of the cell and can not exist in the cell culture medium. From the results, it can be seen that M1 and M2 proteins constitute virus-like particles.

When the 30% sucrose cushion precipitate was observed with an electron microscope, the presence of virus-like particles having a size of about 100 nm was confirmed (FIG. 5).

Using the recombinant vector of Example 1, virus-like particles composed of influenza M1 and M2 proteins were stably produced in the transformed tricofluorocyanine BT1-Tn-5B1-4 cells as described in Example 2 Able to know.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Method for manufacturing influenza vaccine material M1 protein,          M2 protein, and M1-M2 virus-like-particle using insect cell          expression system <130> P13-086-KHU <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 759 <212> DNA <213> Influenza H1N1 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> Influenza H1N1 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> 563 <212> DNA <213> Artificial Sequence <220> <223> Orgyia pseudotsugata immediate-early 2 promoter <400> 3 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> 4 <211> 156 <212> DNA <213> Artificial Sequence <220> <223> Orgyia pseudotsugata immediate-early 2 polya terminator <400> 4 gtttatctga ctaaatctta gtttgtattg tcatgtttta atacaatatg ttatgtttaa 60 atatgttttt aataaatttt ataaaataat ttcaactttt attgtaacaa cattgtccat 120 ttacacactc ctttcaagcg cgtgggatcg atgctc 156 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> pOpIE2 forward primer <400> 5 gatatctcat gatgataaac aatgtatggt gc 32 <210> 6 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pOpIE2 reverse primer <400> 6 gcggccgcaa gctttaaatt cgaacagatg c 31 <210> 7 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> OpIE2 pA forward primer <400> 7 gaattcgttt atctgactaa atcttagttt g 31 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> OpIE2 pA reverse primer <400> 8 gatatcgagc atcgatccca cgcgcttg 28

Claims (9)

A recombinant vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2. 2. The recombinant vector according to claim 1, wherein the recombinant vector further comprises a promoter gene of SEQ ID NO: 3 and a terminator gene of SEQ ID NO: An insect cell transformed with the recombinant vector of claim 1 or 2. The insect cell of claim 3, 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) producing a recombinant vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2; and
(B) an influenza M1 protein, an influenza M2 protein, and an influenza M1-M2 virus-like particle, which comprises the step of transforming an insect cell with the recombinant vector. (A) preparing a recombinant vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2;
(B) transforming the insect cells with the recombinant vector; and
A method for producing influenza M1-M2 virus-like particles, further comprising the step of obtaining influenza M1-M2 virus-like particles (VLP) from a culture medium in which the transformed insect cells have been cultured. (A) preparing a recombinant vector comprising the influenza M1 gene of SEQ ID NO: 1 and the influenza M2 gene of SEQ ID NO: 2;
(B) transforming the insect cells with the recombinant vector; and
(B) obtaining an influenza M1 protein and an M2 protein from the transformed insect cells; and (c) obtaining an influenza M1 protein and an influenza M2 protein. The recombinant vector according to claim 5, wherein the recombinant vector comprises the influenza M1 protein, the influenza M2 protein, and the influenza M1-M2 virus-like particle further comprising the promoter gene of SEQ ID NO: 3 and the terminator gene of SEQ ID NO: &Lt; / RTI &gt; The method according to claim 5, wherein the insect cells of step (b) are selected from the group consisting of BT1-Tn-5B1-4 cells of Trichoplusia ni, LD652Y cells of Lymantria dispar, Spodoptera frugiperda Sf9 cells of Spodoptera frugiperda, Sf21 cells of Spodoptera frugiperda, Kc1 cells of Drosophila, SL2 cells of Drosophila, and mosquito cell lines An influenza M1 protein, an influenza M2 protein, and an influenza M1-M2 virus-like particle.

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