JPWO2015119291A1 - Virus-like particles - Google Patents

Abstract

The present invention is a virus that can be used to produce a vaccine having a higher immunogenicity for preventing infectious diseases in a short period of time, and further producing a vaccine with reduced side effects such as anaphylactic shock and allergic reaction. It is an object to provide like particles, a method for producing the same, and such a vaccine. The present invention is a virus-like particle comprising a recombinant viral membrane protein and a lipid derived from Bombus mori. In the virus-like particle, spikes of the recombinant virus membrane protein are densely arranged on the surface of a hollow spherical outer shell made of the lipid-containing component.

Description

  The present invention relates to a virus-like particle and a method for producing the same, and a vaccine containing the virus-like particle.

  Vaccines are widely used as an effective preventive measure against viral infections. In particular, influenza is a viral infectious disease that is a pandemic around the world every year, so there is a great demand for vaccines. In the influenza causative virus, membrane proteins hemagglutinin (HA) and neuraminidase (NA) are present on the surface of the particles, and viruses are classified according to combinations of subtypes of these membrane proteins. . Although the antigenic subtypes of influenza viruses that are prevalent are determined to some extent, the types that are actually prevalent vary depending on the year, so it is necessary to produce vaccines that match the annual epidemic predictions. Among them, many of the viruses whose HA subtype is H5 or H7 are strongly pathogenic, and so far, many infected people have been produced all over the world, and there have been deaths. Therefore, an effective vaccine is required. ing. Such a vaccine is required to have higher immunogenicity in order to obtain an excellent preventive effect. In addition, to prepare for major epidemics around the world, mass production is possible, low-cost production is possible, production is possible in a short period of time in time for the epidemic, and side effects due to vaccine administration are reduced. What is being done is also required.

  However, traditionally grown chicken eggs of 10 to 11 days are widely used for the production of influenza vaccines. Since it is essential to handle seed viruses for the production of vaccines using these eggs, it is necessary to use highly safe facilities such as the P3 facility, to require a large amount of eggs, and thus to be high in cost. Insufficient fertility, cause egg allergy, long-term production, may change antigenicity to increase in chicken eggs, manufacture component vaccines with only active ingredients There are various disadvantages such as being difficult.

  In addition, today's scientific and technological advances have made it possible to produce influenza vaccines using MDCK or VERO of cultured cells. Of particular interest is the genetic manipulation technology using various vectors, which makes it possible to produce the desired HA protein for influenza vaccine in yeast or silkworm (Bombux moly) individuals or their cultured cells. (See Patent Document 1 and Non-Patent Document 1). However, these methods have solved the problem of side effects of egg allergy and improved the expression efficiency of the protein for influenza vaccine, but the production efficiency is still not sufficient.

International Publication No. 2007/046439

Maeda, S .; et al. , Nature (1985) 315, 592-594.

  In this way, vaccine production using conventional embryonated eggs, cultured cells, etc. is expensive, difficult to mass-produce, requires a long time for production, and the immunogenicity of the resulting vaccine is insufficient There are inconveniences such as being. In addition to the allergies, the vaccines obtained thereby have the problem of significant side effects due to the use of adjuvants such as oil. In order to solve these problems, a new mass production technology for virus membrane proteins for vaccines and a highly immunogenic vaccine produced using the technology are required. In other words, what is expected of a near-future vaccine is that it is a component vaccine of a viral protein that is an essential component of the vaccine, second that it can be mass-produced, and third that it does not require an adjuvant. Fourth, there are no or minimal side effects.

  Therefore, the present invention is a virus-like particle that is used for producing a large amount of a vaccine for preventing viral infection that has higher immunogenicity and reduced side effects such as anaphylactic shock and allergic reaction in a shorter period of time, And a method for producing the same, and to provide such a vaccine.

The invention made to solve the above problems is as follows.
[1] A virus-like particle comprising a recombinant virus membrane protein and a lipid derived from Bombus mori.
[2] The virus-like particle according to [1], wherein spikes of the recombinant virus membrane protein are densely arranged on the surface of a hollow spherical outer shell made of the lipid-containing component.
[3] The virus-like particle according to [1] or [2], wherein the spherical outer shell has a diameter of 30 nm to 300 nm.
[4] The virus-like particle according to any one of [1] to [3], wherein the spike has a length of 5 nm to 30 nm.
[5] The virus-like particle according to any one of [1] to [4], wherein the weight ratio of the recombinant virus membrane protein to the lipid is 1: 1 to 10: 1.
[6] The virus-like particle according to any one of [1] to [5], wherein the recombinant virus membrane protein has an amino acid sequence encoded by a nucleic acid having a sequence codon-optimized for expression in Bombux Mori.
[7] The virus-like particle according to any one of [1] to [6], wherein the recombinant virus membrane protein is a recombinant influenza virus HA protein.
[8] The virus-like particle according to [7], wherein the influenza virus in the recombinant influenza virus HA protein is an influenza A virus.
[9] The virus-like particle according to [8], wherein the influenza A virus is H5 or H7.
[10] The virus-like particle according to any one of [1] to [6], wherein the recombinant virus membrane protein is a recombinant Japanese encephalitis virus protein.
[11] The virus-like particle according to [6], wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs: 3, 11, 14, 17, 20, 23, 26, 29, and 32.
[12] Any of [6] to [10], wherein the codon optimized DNA sequence is selected from the group consisting of SEQ ID NOs: 4, 12, 15, 18, 21, 24, 27, 30, and 33 The virus-like particle according to crab.
[13] The virus-like particle according to any one of [1] to [12], which is used for a vaccine.
[14] A vaccine comprising the virus-like particles according to [1] to [13].
[15] The vaccine of claim 14, which is an oral vaccine.
[16] A step of obtaining a recombinant baculovirus by introducing a recombinant viral membrane protein gene having a sequence codon-optimized for expression in Bombyx Mori into a baculovirus,
Inoculating the above-mentioned recombinant baculovirus into Bombyx Mori moth,
The step of rearing the pupae at 20-30 ° C. for 2-6 days,
A method for producing virus-like particles comprising the steps of producing an emulsion by crushing and emulsifying the pupae after breeding, and obtaining a fraction containing virus-like particles from the emulsion.

  According to the present invention, it has become possible to provide a large amount of a highly immunofunctional vaccine for preventing viral infection that has high immunogenicity and reduced side effects in a short period of time at a low cost. The virus-like particle of the present invention is very similar to a virus particle, and has a structure in which only a recombinant virus membrane protein is tightly bound to the surface of a hollow spherical outer shell made of a lipid-containing component. Compared with significantly higher immunogenicity (immunogenicity). Furthermore, since the virus-like particle of the present invention exhibits high immunogenicity even by oral inoculation, it can be suitably used as an oral vaccine. In addition, because of such high immunogenicity, it is not necessary to use an adjuvant at the time of inoculation, so that side effects such as anaphylactic shock and allergy can be reduced, and the safety is excellent.

The codon usage frequency in Bombux Mori (scientific name: Bombyx mori) is shown. It is the table | surface which investigated the usage frequency in 450043 codon in the 1180 coding region (CDS) of Bombux Mori. The frequency of appearance per 1000 codons is shown. The numbers in parentheses indicate the total number of occurrences. The bold text indicates that it is the most frequently used gene codon for each amino acid. FIG. 3 shows an optimized codon correspondence table based on the most frequently used gene codons shown in FIG. Serine (S) is UCA and its complementary strand is TGA, which is a stop codon. Therefore, the complementary strand of serine serves as a stop codon and no large frame appears in the complementary strand. The correspondence between the avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of HA gene of highly pathogenic avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1), optimization of FLAG tag at the C-terminus, codon usage of Bombux Mori cells The base sequence of the synthetic gene was determined. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of HA gene of highly pathogenic avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) The base sequence of the synthetic gene was determined. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of HA gene of highly pathogenic avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) The base sequence of the synthetic gene was determined. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of HA gene of highly pathogenic avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) The base sequence of the synthetic gene was determined. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. It is the result of the analysis of the coding frame of the gene which codes the HA protein for vaccines synthesize | combined. From the bottom, N1> −, N2> −, and N3> − are the results of analyzing three coding frames of the plus strand, respectively. The upper bar indicates the position of the start codon ATG, and the lower bar indicates the location of the stop codon (TAA, TAG, TGA). Only N1> − is one large coding frame, but in other frames, there are many bars that stick out below, and even if expressed, a large protein cannot be made. N1 <−, N2 <−, N3 <− from the fourth to the top from the bottom are the results of analyzing the coding frame of the complementary strand. Even if any frame is expressed, a large protein cannot be made. The alignment of the codon optimized HA gene DNA sequence with the HA gene cDNA sequence of avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) is shown. Query indicates a codon optimized HA gene DNA sequence (coding region sequence excluding FLAG TAG), and Sbjct indicates avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) (HA coding region sequence). Parts having the same sequence are indicated by *, and-indicates a gap. Although the homology is very low at 77%, it is designed to express the same amino acid sequence except that the highly toxic sequence is modified and deleted. Figure 2 shows an alignment of the codon optimized HA gene DNA sequence with the avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) virus HA gene cDNA sequence. Query indicates a codon optimized HA gene DNA sequence (coding region sequence excluding FLAG TAG), and Sbjct indicates avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) (HA coding region sequence). Parts having the same sequence are indicated by *, and-indicates a gap. Although the homology is very low at 77%, it is designed to express the same amino acid sequence except that the highly toxic sequence is modified and deleted. The expression confirmation by Western blotting of the HA protein produced using Bombux Mori is shown. Arrows indicate specific bands. The HI activity with respect to various influenza viruses of the HI antibody in the chicken by the HA solution produced using pBm-8HA is shown. Reacted extensively with natural viruses. The HA activity of each fraction of the sucrose density gradient is shown. The HA activity was distributed at a lower density position than the position where the natural virus settled. The electron microscope image in a sucrose density gradient fraction is shown. Many virus-like particles having a diameter of 30 to 300 nm were observed. Arrows indicate HA protein spikes that are densely present on the surface of the virus-like particle. The electron microscope image in a sucrose density gradient fraction is shown. Many virus-like particles having a diameter of 30 to 300 nm were observed. Arrows indicate HA protein spikes that are densely present on the surface of the virus-like particle. The electron microscope image in a sucrose density gradient fraction is shown. Many virus-like particles having a diameter of 30 to 300 nm were observed. The arrow indicates the triangular head portion of the HA protein spike that is densely present on the surface of the virus-like particle. The correspondence between the avian influenza virus A / chicken / Sukabumi / 2008 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of the HA gene of avian influenza virus A / chicken / Sukabumi / 2008 (H5N1), considering the introduction of the FLAG tag at the C-terminal and the optimization of codon usage in Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / chicken / Sukabumi / 2008 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of the HA gene of avian influenza virus A / chicken / Sukabumi / 2008 (H5N1), considering the introduction of the FLAG tag at the C-terminal and the optimization of codon usage in Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / chicken / Sukabumi / 2008 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of the HA gene of avian influenza virus A / chicken / Sukabumi / 2008 (H5N1), considering the introduction of the FLAG tag at the C-terminal and the optimization of codon usage in Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / chicken / Sukabumi / 2008 (H5N1) codon optimized HA gene DNA sequence and amino acid sequence is shown. The box indicates the attenuated site and the FLAG tag. Based on the base sequence of the HA gene of avian influenza virus A / chicken / Sukabumi / 2008 (H5N1), considering the introduction of the FLAG tag at the C-terminal and the optimization of codon usage in Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The expression confirmation by Western blotting of the HA protein produced using Bombux Mori is shown. Arrows indicate specific bands. The correspondence between the hepatitis C virus codon optimized Core-E1-E2 fusion protein DNA sequence and amino acid sequence is shown. Core protein: DNA sequence 1 to 573, E1 protein: 574 to 1149, E3 protein: 1150 to 2238, FLAG tag: 2239 to 2262. The correspondence between the hepatitis C virus codon optimized Core-E1-E2 fusion protein DNA sequence and amino acid sequence is shown. Core protein: DNA sequence 1 to 573, E1 protein: 574 to 1149, E3 protein: 1150 to 2238, FLAG tag: 2239 to 2262. The correspondence between the hepatitis C virus codon optimized Core-E1-E2 fusion protein DNA sequence and amino acid sequence is shown. Core protein: DNA sequence 1 to 573, E1 protein: 574 to 1149, E3 protein: 1150 to 2238, FLAG tag: 2239 to 2262. The correspondence between the hepatitis C virus codon optimized Core-E1-E2 fusion protein DNA sequence and amino acid sequence is shown. Core protein: DNA sequence 1 to 573, E1 protein: 574 to 1149, E3 protein: 1150 to 2238, FLAG tag: 2239 to 2262. The correspondence between the hepatitis C virus codon optimized Core-E1-E2 fusion protein DNA sequence and amino acid sequence is shown. Core protein: DNA sequence 1 to 573, E1 protein: 574 to 1149, E3 protein: 1150 to 2238, FLAG tag: 2239 to 2262. The expression confirmation by Western blotting of the Core-E1-E2 fusion protein produced using Bombux Mori is shown. The correspondence between the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) codon optimized HA gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) HA gene, considering the introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells, The base sequence was designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) codon optimized HA gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) HA gene, considering the introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells, The base sequence was designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) codon optimized HA gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) HA gene, considering the introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells, The base sequence was designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) codon optimized HA gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) HA gene, considering the introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells, The base sequence was designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between Japanese encephalitis virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequences of the Japanese encephalitis virus PreM protein gene, M protein gene, and E protein gene, considering the introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM / M protein: DNA sequence 1 to 501; E protein: 502 to 2001; FLAG tag: 2002 to 2025. The correspondence between Japanese encephalitis virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequences of the Japanese encephalitis virus PreM protein gene, M protein gene, and E protein gene, considering the introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM / M protein: DNA sequence 1 to 501; E protein: 502 to 2001; FLAG tag: 2002 to 2025. The correspondence between Japanese encephalitis virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequences of the Japanese encephalitis virus PreM protein gene, M protein gene, and E protein gene, considering the introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM / M protein: DNA sequence 1 to 501; E protein: 502 to 2001; FLAG tag: 2002 to 2025. The correspondence between Japanese encephalitis virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequences of the Japanese encephalitis virus PreM protein gene, M protein gene, and E protein gene, considering the introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells, the base sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. PreM / M protein: DNA sequence 1 to 501; E protein: 502 to 2001; FLAG tag: 2002 to 2025. The correspondence between the rabies virus codon optimized G protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the G protein gene of rabies virus, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the rabies virus codon optimized G protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the G protein gene of rabies virus, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the rabies virus codon optimized G protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the G protein gene of rabies virus, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the rabies virus codon optimized G protein gene DNA sequence and amino acid sequence is shown. A surrounding line shows a FLAG tag. Based on the base sequence of the G protein gene of rabies virus, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between West Nile virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequence of the E protein gene of West Nile virus, the nucleotide sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between West Nile virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequence of the E protein gene of West Nile virus, the nucleotide sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between West Nile virus codon optimized E protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequence of the E protein gene of West Nile virus, the nucleotide sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminus and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between MERS coronavirus codon optimized spike G protein gene DNA sequence and amino acid sequence is shown. Based on the base sequence of the MERS coronavirus spike G protein gene, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between MERS coronavirus codon optimized spike G protein gene DNA sequence and amino acid sequence is shown. Based on the base sequence of the MERS coronavirus spike G protein gene, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between MERS coronavirus codon optimized spike G protein gene DNA sequence and amino acid sequence is shown. Based on the base sequence of the MERS coronavirus spike G protein gene, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between MERS coronavirus codon optimized spike G protein gene DNA sequence and amino acid sequence is shown. Based on the base sequence of the MERS coronavirus spike G protein gene, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between MERS coronavirus codon optimized spike G protein gene DNA sequence and amino acid sequence is shown. Based on the base sequence of the MERS coronavirus spike G protein gene, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between MERS coronavirus codon optimized spike G protein gene DNA sequence and amino acid sequence is shown. Based on the base sequence of the MERS coronavirus spike G protein gene, the base sequence of the synthetic gene was designed in consideration of introduction of a FLAG tag at the C-terminal and optimization of codon usage of Bombux Mori cells. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the foot-and-mouth disease virus codon optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequences of VP4, VP2, VP3, VP1, 2A, and 3C protein genes of foot-and-mouth disease virus, considering the introduction of the FLAG tag at the N-terminal and the optimization of codon usage of Bombux Mori cells, the nucleotide sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the foot-and-mouth disease virus codon optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequences of VP4, VP2, VP3, VP1, 2A, and 3C protein genes of foot-and-mouth disease virus, considering the introduction of the FLAG tag at the N-terminal and the optimization of codon usage of Bombux Mori cells, the nucleotide sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the foot-and-mouth disease virus codon optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequences of VP4, VP2, VP3, VP1, 2A, and 3C protein genes of foot-and-mouth disease virus, considering the introduction of the FLAG tag at the N-terminal and the optimization of codon usage of Bombux Mori cells, the nucleotide sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the foot-and-mouth disease virus codon optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequences of VP4, VP2, VP3, VP1, 2A, and 3C protein genes of foot-and-mouth disease virus, considering the introduction of the FLAG tag at the N-terminal and the optimization of codon usage of Bombux Mori cells, the nucleotide sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The correspondence between the foot-and-mouth disease virus codon optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene DNA sequence and amino acid sequence is shown. Based on the nucleotide sequences of VP4, VP2, VP3, VP1, 2A, and 3C protein genes of foot-and-mouth disease virus, considering the introduction of the FLAG tag at the N-terminal and the optimization of codon usage of Bombux Mori cells, the nucleotide sequence of the synthetic gene Designed. The amino acid sequence predicted from the base sequence of the synthetic gene is also shown. The expression confirmation by Western blotting of recombinant Japanese encephalitis virus protein (PreM-ME fusion protein) expressed in Bm-N cells is shown. Arrows indicate specific bands. Bm-N cells expressing recombinant Japanese encephalitis virus protein (PreM-ME fusion protein) (b, c, d). (A) is an untreated Bm-N cell. (D) is a Bm-N cell expressing a recombinant Japanese encephalitis virus protein (PreM-ME fusion protein) stained with a monoclonal antibody against the fluorescently labeled Japanese encephalitis virus E protein. The electron micrograph of the Japanese encephalitis virus protein (PreM-ME fusion protein) in a sucrose density gradient fraction is shown. The expression confirmation by Western blotting of the recombinant H7 avian influenza virus (A / duck / Korea / A76 / 2010 (H7N7)) HA protein expressed in Bm-N cells is shown. Arrows indicate specific bands. Recombinant H7 avian influenza virus (A / duck / Korea / A76 / 2010 (H7N7)) Bm-N cells expressing HA protein (b, c, d). (A) is an untreated Bm-N cell. (D) is a Bm-N cell expressing a recombinant H7 avian influenza virus (A / duck / Korea / A76 / 2010 (H7N7)) HA protein stained with a fluorescently labeled guinea pig antiserum against the HA protein. .

  Hereinafter, the present invention will be described in detail.

<Virus-like particles>
The virus-like particle of the present invention comprises a recombinant of a membrane protein of a virus that causes infection, and a lipid derived from Bombus mori. Thus, the virus-like particle | grains of this invention can be equipped with higher immunogenicity by being the structure similar to the virus particle which causes an infectious disease. Therefore, it is possible to achieve a sufficient infection prevention effect without using an adjuvant required for a normal vaccine.

  As the structure of the virus-like particle of the present invention, it is preferable that spikes of the recombinant virus membrane protein are densely arranged on the surface of a hollow spherical outer shell composed of the lipid-containing component. Thus, the virus-like particle | grains of this invention can be equipped with a still higher immunogenicity by having a structure very similar to the virus particle which causes an infectious disease. Hereinafter, the recombinant virus membrane protein and the lipid derived from Bombux moly contained in the virus-like particle of the present invention will be described.

[Recombinant virus membrane protein]
The recombinant viral membrane protein contained in the virus-like particle of the present invention is a recombinant of the membrane protein of the virus causing the viral infection. Examples of the virus causing the virus infection include influenza virus, hepatitis C virus, Japanese encephalitis virus, rabies virus, West Nile virus, MERS coronavirus, foot-and-mouth disease virus and the like.

  Influenza viruses include type A, type B and type C influenza viruses. Among these, from the viewpoint of the effectiveness of the vaccine using the virus-like particle of the present invention and the demand for the vaccine, type A and type B influenza viruses are preferable, and type A influenza virus is more preferable. Among these, H5 type and H7 type are more preferable, and avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) is particularly preferable. For the same reason, genotypes 1a, 1b, 2a, 2b, and 3a are preferred for hepatitis C virus.

  The viral membrane protein is not particularly limited as long as it is an effective protein as a vaccine. For example, in the case of influenza virus, hemagglutinin (HA) protein and neuraminidase (NA) protein can be mentioned. From the viewpoint of effectiveness as a vaccine, HA protein is preferable. In the case of hepatitis C virus, E1 protein and E2 protein are mentioned. In the case of Japanese encephalitis virus, E protein may be mentioned. In the case of rabies virus, G protein can be mentioned. In the case of West Nile virus, E protein may be mentioned. In the case of MERS coronavirus, spike G protein is mentioned. In the case of foot-and-mouth disease virus, VP4, VP2, VP3, VP1, 2A, 3C proteins and the like can be mentioned.

  A plurality of the above-mentioned virus membrane proteins may be contained in one virus-like particle. For example, viral membrane proteins include influenza virus HA protein, hepatitis C virus E1 and E2 proteins, Japanese encephalitis virus E protein, rabies virus G protein, West Nile virus E protein, MERS coronavirus spike One or more combinations selected from the group consisting of G protein and VP4, VP2, VP3, VP1, 2A, and 3C proteins of foot-and-mouth disease virus may be used.

  When the viral membrane protein is an HA protein, the virus-like particles of the present invention preferably do not contain other constituent proteins of influenza virus, such as M1 and M2 proteins.

  The recombinant viral membrane protein is preferably a recombinant of the viral membrane protein described above, and has an amino acid sequence encoded by a nucleic acid having a sequence codon optimized for expression in Bombux Mori. Specifically, the codon optimization uses the amino acid sequence and gene sequence correspondence table (FIG. 2) created based on Bombus Mori's codon usage (FIG. 1) to replace the target nucleic acid sequence. Is done. In this correspondence table, in order to prevent unexpected by-products from being synthesized due to the formation of a long coding frame in the complementary strand, the Ser sequence is complemented using UCA (most frequently used in Bombux Mori). It is designed to produce many UGAs (stop codons) in the chain.

  Moreover, the nucleic acid encoding the recombinant virus membrane protein may have an attenuated gene modification. Attenuation can be accomplished by any modification known to those skilled in the art. For example, in the case of the HA protein of influenza virus, the sequence of the cleavage site that joins HA1 and HA2 molecules that control pathogenicity is modified. Specifically, the amino acid sequence represented by SEQ ID NO: 7 is substituted with the amino acid sequence represented by SEQ ID NO: 8 in the HA protein.

  The nucleic acid encoding the recombinant viral membrane protein is preferably a nucleic acid comprising or consisting of the sequences of SEQ ID NOs: 4, 12, 15, 18, 21, 24, 27, 30, or 33.

  It is preferable that the recombinant virus membrane protein contained in the virus-like particle of the present invention is densely arranged as a spike on the surface of a hollow spherical outer shell made of a component containing lipids derived from Bombux Mori.

  The length of the spike is preferably 5 nm to 30 nm, more preferably 8 nm to 25 nm, still more preferably 10 nm to 20 nm, and particularly preferably 12 nm to 18 nm. When the recombinant virus membrane protein spike has a length in the above-mentioned range, the virus-like particle of the present invention has a structure more similar to a virus particle, and can improve immunogenicity when used in a vaccine. it can.

  The spike has a wedge shape with a triangular head portion, and the end portion can be bonded to a spherical outer shell made of a component containing a lipid derived from Bombus moly.

[Limb from Bombux Mori]
The Bombux Mori derived lipid contained in the virus-like particle of the present invention is not particularly limited as long as it is a lipid possessed by Bonbux Mori, and includes lipids possessed by Bombux Mori cells, Bombux Mori larvae, pupae and the like. It is done.

  Examples of the lipid include glyceroglycolipid, glycosphingolipid, cholesterol, phospholipid and the like.

  Examples of the glyceroglycolipid include sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.

  Examples of the glycosphingolipid include galactosyl cerebroside, lactosyl cerebroside, ganglioside and the like.

  Examples of the phospholipid include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, lysophosphatidylcholine, sphingomyelin and the like.

  Fatty acids derived from the above lipids by hydrolysis or the like may also be included. Examples of the fatty acid include myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linolenic acid, and the like.

  In the virus-like particle of the present invention, it is preferable that these components containing lipids derived from Bombux-Mori form a hollow spherical outer shell. That is, the spherical outer shell has a virus envelope-like structure, and is more preferably a lipid bilayer composed of the lipid and the like.

  The diameter of the spherical outer shell is preferably 30 nm to 300 nm, more preferably 50 nm to 200 nm, still more preferably 70 nm to 150 nm, and particularly preferably 80 nm to 120 nm.

  The weight ratio of the recombinant virus membrane protein and the lipid is 1: 1 to 10: 1.

  The virus-like particle of the present invention has a structure in which the surface of the spherical outer shell composed of the lipid-containing component is densely covered with the spike of the recombinant virus membrane protein. Thus, the vaccine containing the virus-like particle of the present invention has a structure with closely packed spikes of recombinant virus membrane protein with immunogenicity, so that there is no conventional one without using an adjuvant. It becomes possible to show high immunogenicity. Since no adjuvant is used, side effects such as allergies and anaphylactic shock can be reduced. Of course, the virus-like particles of the present invention are non-pathogenic because no virus-derived nucleic acid is present in the hollow shell of the spherical outer shell. In addition, the hollow of the spherical outer shell may contain physiological saline, phosphate buffer, or the like.

  The virus-like particle of the present invention may contain components derived from Bombus moly other than lipids, such as proteins and sugars derived from Bonbucus moly. Preferably, the virus-like particle of the present invention is substantially free of components derived from Bombux Mori other than lipids.

[Method for producing virus-like particles]
The virus-like particle of the present invention can be produced by the following method. That is,
A step of obtaining a recombinant baculovirus by introducing a recombinant viral membrane protein gene having a sequence codon-optimized for expression in Bombux Mori into a baculovirus;
Inoculating the above-mentioned recombinant baculovirus into Bombyx Mori moth,
The step of rearing the pupae at 20-30 ° C. for 2-6 days,
It is a method for producing virus-like particles, comprising the steps of producing an emulsion by crushing and emulsifying the pupae after breeding, and obtaining a fraction containing virus-like particles from the emulsion.

  The recombinant viral membrane protein gene is introduced into Bombux Mori using a recombinant baculovirus into which the recombinant viral membrane protein gene has been introduced. Introduction of a recombinant viral membrane protein gene into a baculovirus vector can be performed by any method known to those skilled in the art. The time of introduction (inoculation) of the recombinant baculovirus into Bonbucus Mori is not particularly limited as long as it is in the last stage, but it is preferably early in the last stage, specifically, on the day of the first stage (Day 1 of molting) to 4th day (day 5 of molting) are preferred, and the next day (day 2 of molting) to 2 days after molting (day 3 of molting) are more preferred.

  After inoculating the above-mentioned recombinant baculovirus in Bombyx moly pupae, these pupae are bred for a predetermined period, and the temperature condition is preferably 20 ° C. to 30 ° C., more preferably 22 ° C. to 28 ° C. 24 ° C to 27 ° C is more preferable, and 26 ° C is particularly preferable. The breeding period is not particularly limited as long as it is a period from the inoculation to before the pupae are transformed into adults (moths), but preferably 2 to 6 days after the inoculation, more preferably 2 to 4 days. More preferably, 3 days.

  Recovery of recombinant viral membrane proteins from Bombux Mori can be done using any method known to those skilled in the art. For example, when expressing recombinant HA protein of influenza virus, homogenize Bonbux moly sputum in isotonic buffer solution, and then recover using immobilized red blood cells or sialic acid column (fetuin column) can do. In addition, a fraction containing recombinant virus membrane protein in the form of virus-like particles can be obtained using a fractionation method such as sucrose density gradient centrifugation.

[vaccine]
The vaccine of the present invention contains the virus-like particle of the present invention. Since the virus-like particle of the present invention has high immunogenicity, it is possible to exhibit high immunogenicity that has not existed before without using an adjuvant. Since no adjuvant is used, side effects such as allergies and anaphylactic shock can be reduced. Of course, the virus-like particles of the present invention are non-pathogenic because no virus-derived nucleic acid is present in the hollow interior of the spherical outer shell. The vaccine of the present invention can effectively prevent virus infection of animals including humans. Moreover, the vaccine of this invention can shorten the period required for manufacture remarkably compared with the manufacturing method etc. which used the conventional egg. As a result, vaccines can be procured in a short period of time during the epidemic of viral infections such as influenza, which can greatly contribute to protecting people from infections and maintaining their health. Furthermore, in the production of vaccines using conventional eggs, large-scale facilities such as P3 facilities are required to handle seed viruses used in vaccines, and a large amount of vaccines are produced due to low vaccine immunogenicity. The manufacturing method of the present invention can remarkably reduce the manufacturing cost, while enormous manufacturing cost is necessary due to the necessity.

  An effective amount of vaccine refers to an amount sufficient to achieve a biological effect such as inducing sufficient humoral or cellular immunity against the virus. Administration methods also include inhalation, intranasal, oral, parenteral (eg, intradermal, intramuscular, intravenous, intraperitoneal, and subcutaneous administration). The effective amount and method of administration may depend on the age, sex, condition, weight of the human being administered. For example, in the case of influenza vaccine, a vaccine containing 15 μg or more of HA protein in 1 ml is generally administered, 0.25 ml is subcutaneously administered to those who are 6 months or older and younger than 3 years, and 0 to 3 years or older and younger than 13 years. Inject 5 ml subcutaneously twice, approximately 2-4 weeks apart. For those 13 years and older, 0.5 ml is injected subcutaneously once or twice at approximately 1 to 4 week intervals.

  Hereinafter, the present invention will be described in more detail with specific experimental examples.

[Example 1] Production of virus-like particles (influenza vaccine)
(1) Nucleic acid sequence design of codon-optimized HA gene Gene information of hemagglutinin (HA) protein of highly pathogenic avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) discovered in wild ducks in 2011 The design was changed as follows.

  First, the amino acid sequence (SEQ ID NO: 2) of the hemagglutinin (HA) protein of avian influenza virus A / tufted duck / Fukushima / 16/2011 (H5N1) is obtained from Genbank (URL: http: //www.ncbi.nlm.nih. gov / genbank /) Accession No. Predicted from the gene sequence registered in BAK24078 (SEQ ID NO: 1).

Next, the amino acid sequence predicted, Arg-Glu- Arg is presumed to be related to a highly pathogenic - Arg - Lys - Arg sequence (SEQ ID NO: 7) Arg-Glu- Thr - Arg sequence (SEQ ID NO: 8) And the FLAG tag sequence (SEQ ID NO: 5) consisting of Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys was added to the C-terminus, and the attenuated HA protein amino acid sequence (SEQ ID NO: 3) was added. Designed.

  Codon optimization was performed for the purpose of improving protein expression using Bombux Mori. Bonbux Mori's codon usage (URL: http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=7091) obtained from Codon Usage Database provided by Kazusa DNA Research Institute (Figure) Based on 1), a correspondence table (FIG. 2) of the prepared amino acid sequences and gene sequences was prepared. In this correspondence table, in order to prevent unexpected by-products from being synthesized due to the formation of a long coding frame in the complementary strand, the Ser sequence is UCA (most frequently used in Bombux Mori), and the complementary strand is used. Were designed to generate many UGAs (stop codons). The amino acid sequence corresponding to the designed gene sequence (SEQ ID NO: 4) is shown in FIG. It was confirmed that a large coding frame was not possible except for the protein to be expressed (FIG. 4). The homology between the codon optimized HA and the original HA was 77.5% in the nucleic acid sequence.

(2) Codon-optimized HA gene synthesis and vector construction Based on the sequence information of the designed codon-optimized HA gene, a total synthesis of DNA was requested from Takara Bio Inc. The synthesized codon-optimized HA gene DNA was inserted into pBm-8 (Baculo Technology), which is a transfer vector, using the In-Fusion method (Clontech) to prepare pBm-8H5HA.

(3) Infection of codon-optimized HA gene-introduced baculovirus into Bonbux moly and preparation of emulsions Simultaneous introduction of pBm-8H5HA and DNA extracted from baculovirus into Bonbux moly cells (Bm-N cells) Thus, a recombinant baculovirus for vaccine production was obtained in the cell supernatant. This recombinant baculovirus (H5HA-BmNPV) was inoculated into Bombus mori moths. That is, a virus stock solution having a virus immunogenicity of 1 × 10 ⁸ pfu / ml or more was diluted 10 times with insect cell culture medium TC-100. 50 μl of this diluted solution was inoculated by injection into the silkworm abdomen on the second day of molting, and then the pupae were raised in a 26 ° C. incubator. After 96 hours, the cocoon was cut in half with scissors on ice and the midgut was removed with tweezers, followed by crushing and emulsification in PBS containing phenylthiouric acid using a potter type homogenizer. PBS was added to the emulsion so as to be 44 ml, and 5 μl of formalin was further added so that the final concentration was 0.01%.

(4) Recovery of HA protein The obtained Bombux-Morii cocoon emulsion was sonicated with a sonicator (UR-20P, manufactured by Tommy Seiko) for 5 minutes.

(5) Evaluation of HA activity Influenza virus has the property of agglutinating when mixed with erythrocytes of various animals. This is called hemagglutination, and hemagglutinin (HA) on the surface of the virus binds to the glycans of erythrocytes. By making a large aggregate by cross-linking a plurality of red blood cells. By using this property, the virus concentration (HA activity) contained in the stock solution can be calculated by investigating how far the virus aggregates when serially diluted, so it is used for quantification of influenza virus. Specifically, using a 96-well microplate, 50 μl of the sample was serially diluted with 50 μl of PBS (phosphate buffered saline), and then mixed with equal amounts of chicken erythrocytes adjusted to 0.5%. And let stand at room temperature for 30-60 minutes. If the subject has HA activity, an agglomerate is formed with erythrocytes, but if no virus is present, the agglomerate cannot be formed and sinks like a Japanese circle at the bottom of the plate. The HA value as the HA aggregation activity is expressed by the magnification of the dilution point before the day circle is formed.

  The obtained HA protein solution was evaluated under the above conditions. As a result, the HA activity was 2,097,152. As a result, the total HA activity produced in 220 Bombux mori moths was 2,097,152 × 44 ml = 92,274,688, and the amount of HA activity per moth was 419,430. This value of the amount of HA activity in one recombinant Bombyx Mori is about 205 times to 410 times the HA activity of 1,024 to 2,048 produced from a single hen's egg. An increase in quantity could be realized.

(5) Immunization of chicken with HA protein solution (i) Administration by intravenous injection and intramuscular injection In order to confirm the effect of the HA protein solution as a vaccine, chickens were immunized with the HA protein solution. 0.5 ml of HA protein solution with HA activity adjusted between 8,192 and 16,384 was intravenously administered to 1 month old female chickens, and the same amount to chickens on days 16 and 33 The leg muscle was inoculated. On the first inoculation day, blood was collected from chickens 16 days, 33 days and 40 days after the initial inoculation.
(Ii) Oral administration In order to confirm the effect of the HA protein solution as a vaccine, mice were immunized with the HA protein solution. 0.5 ml of HA protein solution with HA activity adjusted between 8,192 and 16,384 was administered twice and three times to five 4 week old mice (ddY, sputum). In the case of two administrations, oral administration was carried out 14 days after the first administration on the day of the first inoculation, and blood was collected from the mice 28 days after the first administration. In the case of three administrations, oral administration was carried out 14 days after the first administration, 21 days after the first administration, and blood was collected from the mice 28 days after the first administration.

(6) Evaluation of HI activity of antibody produced by immunized chicken (i) Administration by intravenous injection and intramuscular injection Each of the above-collected chicken blood is allowed to stand at room temperature and then centrifuged at room temperature to obtain chicken serum. Got. The erythrocyte aggregation inhibitory activity (HI activity) of each chicken serum was examined by the following method. First, using a 96-well plate, a 1/2 serial dilution series of serum containing 25 μl of each well was prepared. Next, 25 μl of the HA protein (prepared to 8 HA with PBS) was added to each well. After allowing the plate to stand at room temperature for 30 minutes, 50 μl of 0.5% / PBS chicken erythrocytes were added to each well. One hour later, the erythrocyte aggregation pattern was observed, and the maximum dilution factor at which erythrocyte aggregation was observed was defined as the HI activity value. Sera 20 days after the first inoculation showed 128 HI activity and sera 33 days later showed 8,192 HI activity.
(Ii) Oral administration Mouse serum was obtained by allowing the blood of the collected mice to stand at room temperature and then centrifuging at room temperature. The hemagglutination inhibitory activity (HI activity) of each mouse serum was measured by the same method as in the case of the chicken serum. The serum 21 days after the third administration showed a HI activity of 128-1624, confirming a significant increase in antibody production. It was revealed that the HA protein (virus-like particle) of the present invention sufficiently functions as an oral vaccine. That is, the HA protein (virus-like particle) of the present invention can maintain immunogenicity even when exposed to gastric juice conditions, and the mouse serum after three oral inoculations will eventually have high HI activity. Indicated. Table 1 below shows the antibody titers (HI activity) of mouse serum after oral administration of HA protein (virus-like particles) three times.

(7) Evaluation of HI activity against various influenza viruses Using chicken serum 40 days after the first inoculation, the presence or absence of HI activity against various influenza viruses was examined. A / duck / Singapore-Q / F119-3 / 1997 (H5N3), A / chicken / Legok / 2004 (H5N1), A / chicken / West Java / 2009 (H5N1), Silkworm-derived (in this example) HI activity was measured using the prepared HA protein) under the same conditions as described above. The results are shown in FIG. Chicken serum showed HI activity against all viruses.

(8) Fractionation of HA protein solution by sucrose density gradient (fractionation of virus-like particles)
The HA protein solution was fractionated by the sucrose density gradient method. The HA activity of each fraction was examined by the same method as described above. The result is shown in FIG. The fraction with a buoyant density of 1.18 g / l of the native virus showed no HA activity. On the other hand, HA activity was observed in fractions 20 to 31.

(9) Observation of HA protein solution fraction by electron microscope (observation of virus-like particles)
The sample which pooled fractions 20-31 of said sucrose density gradient was observed with the electron microscope. The result is shown in FIG. A virus particle-like structure having a spherical shape with HA protein spikes with a length of about 14 nm (average value) on the surface and a particle diameter of 30 nm to 300 nm was observed. The spike density of the HA protein in this example was higher than the spike density on influenza virus particles. Moreover, as shown in FIG. 9, the spike of the HA protein has a wedge shape with a triangular head portion, suggesting that it forms a trimer in the same manner as HA present on an actual virus. This morphological feature is considered to be reflected in the above strong immunity that was not found in conventional vaccines.

(10) Confirmation of HA protein by Western blotting analysis The sample of fraction 25 of the above sucrose density gradient was subjected to Western blotting (primary antibody: anti-FLAG mouse monoclonal antibody, secondary antibody: anti-mouse IgG rabbit polyclonal antibody). The HA protein having a molecular weight of 73.1 KD was confirmed (FIG. 6).

(11) Component analysis of HA protein solution fraction (component analysis of virus-like particles)
With respect to the HA protein solution fraction obtained from the sucrose density gradient, the amount of protein and the amount of lipid were measured according to a conventional method. As a result, the protein concentration was 0.75 mg / ml, and the lipid concentration was 0.28 mg / ml.

  As described above, the activity level of the HA protein of avian influenza virus A / tufted duck / Fukushima / 16/2011 increased due to Bonbux Mori's changes in DNA design based on genetic information. It is thought that the genetic manipulation of the synthetic DNA based on it worked beneficially. Furthermore, since the target gene was highly expressed using the bud stage of Bombus mori, it is considered that the obtained vaccine exhibited a virus particle-like structure. In this way, with the background of genetic information, new vaccine production methods utilizing DNA design changes and synthesis are expected to be used in multiple fields in the future.

[Example 2] Influenza vaccine Amino acid sequence information (SEQ ID NO: 10) predicted from the HA gene sequence (SEQ ID NO: 9) of avian influenza virus A / chicken / Sukabumi / 2008 (H5N1) isolated in Indonesia in 2008 Based on this, DNA for vaccine development for influenza virus was designed in the same manner as in Example 1 (SEQ ID NO: 12). The corresponding amino acid sequence (SEQ ID NO: 11) is shown in FIG. In the same manner as in Example 1, the recombinant baculovirus containing the above-mentioned development DNA was taken into Bombux mori cage, HA protein (virus-like particles) was synthesized and recovered, and expression was confirmed by Western blot ( FIG. 11). When HA activity was evaluated, the amount of HA activity per rabbit was 419,430. In addition, the HA protein can be fractionated in the same manner as in Example 1, and the component analysis of virus-like particles and the confirmation of virus-like particles by an electron microscope can be performed.

[Example 3] Hepatitis C virus vaccine A DNA for hepatitis C virus vaccine development suitable for production in Bombux Mori was designed as follows. That is, in order to develop a hepatitis C virus vaccine, gene information for the fusion protein expression of the hepatitis C virus core protein-E1 protein-E2 protein was designed. The fusion protein was designed here because it was expected that a virus particle-like protein could be synthesized by simultaneous expression. First, the FLAG tag sequence (SEQ ID NO: 5) is added to the amino acid sequence (SEQ ID NO: 13) from the Core protein to the E1 protein and E2 protein in the amino acid sequence of the HCV gene registered in Genbank under Accession No. ACK28185. In addition, the amino acid sequence of the fusion protein that serves as the basis for nucleic acid design was obtained (SEQ ID NO: 14). Based on the amino acid sequence of this fusion protein, the nucleic acid sequence of the codon-optimized Core-E1-E2 fusion protein was designed in the same manner as in Example 1 (SEQ ID NO: 15). FIG. 12 shows the correspondence between the nucleic acid sequence and amino acid sequence of the designed chimeric synthetic DNA.

  Based on the designed nucleic acid sequence of the codon-optimized Core-E1-E2 fusion protein gene, a full-length gene DNA was synthesized in the same manner as in Example 1 and inserted into the pBm-8 vector to create pBM-8Core-E1-E2. did.

  Recombinant baculovirus was obtained using pBm-8Core-E1-E2 in the same manner as in Example 1, and inoculated into Bonbux Mori cocoons to obtain Bonbux Mori mulberry emulsion. After sonication of the obtained Bombux-Morii cocoon emulsion with a sonicator (UR-20P, manufactured by Tommy Seiko) for 5 minutes, sucrose density gradient centrifugation (sucrose concentration 10-50%, 100,000 G, 24 hours) ) To purify the Core-E1-E2 fusion protein. The purified Core-E1-E2 fusion protein was confirmed by Western blotting. As shown in FIG. 13, a band was confirmed around 60 kDa. In addition, an immune reaction can be predicted from the amount of protein produced, and it is possible to estimate the effect in a neutralization test using the antibody produced thereby.

  Assuming that the Core-E1-E2 fusion protein is expressed in a linked manner, it is expected to show a molecular weight of 83 kDalton or higher, and thus the molecular size of 60 kDa is matured by processing Core-E1-E2 by protease in the cell. It was suggested that E2 was synthesized. This fact shows that the vaccine protein designed without any doubt was produced, and it can be seen from the density of the protein band that the vaccine for hepatitis C was efficiently synthesized. In addition, the Core-E1-E2 fusion protein can be fractionated in the same manner as in Example 1, and component analysis of virus-like particles can be performed, and virus-like particles can be confirmed with an electron microscope.

[Example 4] Design of DNA for vaccine development of avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) and production of vaccine

  GenBank Accession No. Similar to Example 1 based on the amino acid sequence information (SEQ ID NO: 16) of the HA gene sequence of avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) isolated in Korea registered in JN244245 in 2010 A DNA for influenza vaccine development was designed (SEQ ID NO: 18). The corresponding amino acid sequence (SEQ ID NO: 17) is shown in FIG. In the same manner as in Example 1, a baculovirus recombinant into which DNA for influenza virus vaccine development was incorporated was obtained and infected with silkworm Bm-N cells. Western blotting was performed to confirm the expression of recombinant H7 avian influenza virus (A / duck / Korea / A76 / 2010 (H7N7)) HA protein in Bm-N cells. The results are shown in FIG. Specific bands are indicated by arrows. The infected Bm-N cells were stained with guinea pig antiserum against H7 avian influenza virus (A / duck / Korea / A76 / 2010 (H7N7) HA). It was confirmed that the protein was expressed (FIGS. 22-2, d) In addition, as in Example 1, the recombinant baculovirus containing the above-described developmental DNA was ingested into a Bombux Mori cocoon. The HA protein was synthesized and recovered to obtain virus-like particles, and the HI activity of the virus-like particles was evaluated to confirm the effectiveness of the HA protein as a vaccine. Virus-like particles) were immunized to mice, ie 0.5 ml of HA protein solution with HA activity adjusted between 4,092 and 8,192. The virus-like particles), 4-week-old (ddY, in 5 mice of ♀), blood was collected from a total of 2 times orally administered. Mice 28 days after the first dose at two-week intervals.

Each of the collected mouse blood was allowed to stand at room temperature and then centrifuged at room temperature to obtain mouse serum. The hemagglutination inhibitory activity (HI activity) of each mouse serum was measured by the same method as in the case of the chicken serum. The serum 14 days after the second administration showed HI activity of 64-256, and a significant antibody titer could be confirmed. It was revealed that the HA protein (virus-like particle) of the present invention sufficiently functions as an oral vaccine. That is, the HA protein (virus-like particle) of the present invention can maintain immunogenicity even when exposed to gastric juice conditions, and the mouse serum after two oral inoculations finally shows high HI activity. It was. The results are shown in Table 1 above.
In addition, the HA protein was fractionated in the same manner as in Example 1, and component analysis of virus-like particles and confirmation of virus-like particles with an electron microscope were performed. The same results as in Example 1 were obtained.

[Example 5] Design of DNA for vaccine development of Japanese encephalitis virus and production of vaccine Japanese encephalitis virus is an encephalitis virus mediated mainly by Culex mosquito, and is currently prevalent in Southeast Asia, India and China. In order to prevent this, a mouse brain infected with the same virus has been used as a vaccine. Recently, a virus for a vaccine in a cultured cell has been cultured. However, new vaccine development is required in order to enhance the immunity of vaccines, reduce side effects, and reduce production costs, and there are great expectations for quality improvement.

  Therefore, the present inventors designed genetic information for expression of E protein of Japanese encephalitis virus in order to develop a Japanese encephalitis virus vaccine. First, the FLAG tag sequence (SEQ ID NO: 5) is added to the amino acid sequence (SEQ ID NO: 19) from the preM protein in the amino acid sequence registered in Genbank under Accession No. ABQ52691 to the M protein and E protein. The amino acid sequence of the Japanese encephalitis virus PreM-ME fusion protein, which is the basis of the design, was obtained (SEQ ID NO: 20). Based on the amino acid sequence of this Japanese encephalitis virus PreM-ME fusion protein, the nucleic acid sequence of the Japanese encephalitis virus codon optimized PreM-ME fusion protein was designed in the same manner as in Example 1 (SEQ ID NO: 21). . FIG. 15 shows the correspondence between the nucleic acid sequence and amino acid sequence of the designed chimeric synthetic DNA. Based on the nucleic acid sequence of the designed Japanese encephalitis codon optimized PreM-ME fusion protein gene, full-length gene DNA was synthesized and inserted into a pBm-8 vector to create pBM-8JevE. A baculovirus recombinant was obtained using pBm-8JevE in the same manner as in Example 1, and this was infected with silkworm Bm-N cells. In order to confirm the expression of the PreM-ME fusion protein in Bm-N cells, Western blotting was performed. The results are shown in FIG. A band of PreM-ME protein was confirmed around 70 kD. Further, when the infected Bm-N cells were stained with a fluorescently labeled monoclonal antibody against JEV E protein, it was clearly stained and it was confirmed that the PreM-ME fusion protein was expressed in the cells. (FIG. 20-2, d).

  The baculovirus recombinant was inoculated into Bombyx Mori cocoons. On the 4th day after inoculation, Bombux-Morii emulsion was obtained. After sonicating the obtained Bombux-Morii strawberry emulsion with a sonicator (UR-20P, manufactured by Tommy Seiko) for 5 minutes, sucrose density gradient centrifugation (sucrose concentration 10-50% (w / w), 100 PreM-ME fusion protein was purified by 2,000 G, 2 hours). The purified PreM-ME fusion protein was confirmed by Western blotting. Further, when the fraction of the obtained PreM-ME fusion protein was observed with an electron microscope, many virus-like particle structures were observed as shown in FIG.

[Reference Example 1] Design of DNA for Rabies Virus Vaccine Development Rabies virus is distributed globally, causing many death hazards, and there is an international demand for the development of an effective, safe and inexpensive vaccine. The current situation is not progressing slowly. Therefore, if a vaccine with strong immunity, safety and low cost is developed, the needs are considered to be global. Currently used vaccines are derived from the brains of rabbits, goats and mice, and in addition, human diploid cells and chicken embryo cells are used. In the future vaccine development, it is desired to develop a component vaccine that can be used at low cost in terms of supply amount and cost, and if the component vaccine is successfully developed using silkworms, its use will be global.

  In order to develop a rabies virus vaccine, genetic information for G protein expression of rabies virus was designed. First, the FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence registered in Genbank under Accession No. ABX46657 (SEQ ID NO: 22) to obtain the amino acid sequence of the rabies virus G protein that serves as the basis for nucleic acid design. (SEQ ID NO: 23). Based on the amino acid sequence of this rabies virus G protein, the nucleic acid sequence of the rabies virus codon optimized G protein was designed in the same manner as in Example 1 (SEQ ID NO: 24). FIG. 16 shows the correspondence between the nucleic acid sequence and amino acid sequence of the designed chimeric synthetic DNA. Based on the nucleic acid sequence of the designed rabies virus codon-optimized G protein gene, a full-length gene DNA is synthesized in the same manner as in the Examples, and inserted into a pBm-8 vector to prepare pBM-8rvG. A baculovirus recombinant is obtained using pBm-8rvG in the same manner as in Example 1, and inoculated into Bonbux Mori cocoons to obtain Bonbux Mori cocoon emulsion. The resulting Bonbux-Mori straw emulsion is produced by sonication in the same manner as in the Examples. The expression of the purified rabies virus G protein is confirmed by Western blotting in the same manner as in Example 1. From the amount of protein produced, an immune reaction can be predicted, and the effect in a neutralization test with the antibody produced thereby can be estimated. Moreover, the G protein of rabies virus can be fractionated in the same manner as in Example 1, and the component analysis of virus-like particles and the confirmation of virus-like particles by an electron microscope can be performed.

[Reference Example 2] Design of DNA for West Nile virus vaccine development West Nile virus has spread to the United States, Eastern Europe, and Europe, and is likely to be transmitted to Japan in the near future. In addition to the Carterica ecosystem, it is transmitted from this cycle to humans and to horses. There is no vaccine available yet, but the development of this vaccine is urgent worldwide. Research and development of vaccines is actively conducted in the United States and Europe, but has not been successful. Therefore, if West Nile fever vaccine can be produced in large quantities at low cost, it can be used worldwide.

  In order to develop a West Nile virus vaccine, genetic information for the expression of West Nile virus PreM-ME fusion protein is designed. First, a FLAG tag sequence (SEQ ID NO: 5) is added to the amino acid sequence (SEQ ID NO: 25) from the preM protein in the amino acid sequence registered in Genbank under Accession No. AAT95390 to the M protein and E protein. The amino acid sequence of the West Nile virus PreM-ME fusion protein, which is the basis of the design, was obtained (SEQ ID NO: 26). Based on the amino acid sequence of this West Nile virus PreM-ME fusion protein, the nucleic acid sequence of West Nile virus codon optimized PreM-ME fusion protein was designed in the same manner as in Example 1 (SEQ ID NO: 27). . FIG. 17 shows the correspondence between the nucleic acid sequence and amino acid sequence of the designed chimeric synthetic DNA. Based on the nucleic acid sequence of the designed West Nile virus codon-optimized PreM-ME fusion protein gene, the full-length gene DNA is synthesized in the same manner as in the Examples, and inserted into the pBm-8 vector to prepare pBM-8wnvpMME. A baculovirus recombinant is obtained using pBm-8wnvpMME in the same manner as in Example 1, and inoculated into Bonbux Mori cocoons to obtain Bonbux Mori emulsion. The resulting Bombux-Morri cocoon emulsion was produced by sonication in the same manner as in the Examples. Expression of the purified West Nile virus PreM-ME fusion protein can be confirmed by Western blotting in the same manner as in the Examples. From the amount of protein produced, an immune reaction can be predicted, and the effect in a neutralization test with the antibody produced thereby can be estimated. In addition, the West Nile virus PreM-ME fusion protein can be fractionated in the same manner as in Example 1, and component analysis of virus-like particles can be performed, and virus-like particles can be confirmed with an electron microscope.

Reference Example 3 Design of DNA for MERS Coronavirus Vaccine Development In order to develop a MERS coronavirus vaccine, gene information for the expression of spike G protein of MERS coronavirus is designed. First, the FLAG tag sequence (SEQ ID NO: 5) is added to the amino acid sequence (SEQ ID NO: 28) registered in Genbank under Accession No. AGN52936 to obtain the amino acid sequence of West Nile virus E protein that serves as the basis for nucleic acid design. (SEQ ID NO: 29). Based on the amino acid sequence of the MERS coronavirus spike G protein, the nucleic acid sequence of the MERS coronavirus codon optimized spike G protein was designed in the same manner as in Example 1 (SEQ ID NO: 30). FIG. 18 shows the correspondence between the nucleic acid sequence and amino acid sequence of the designed chimeric synthetic DNA. Based on the nucleic acid sequence of the designed MERS coronavirus codon-optimized spike G protein gene, the full-length gene DNA is synthesized in the same manner as in the Examples, and inserted into the pBm-8 vector to prepare pBM-8mcvsG. In the same manner as in Example 1, pBm-8mcvsG is used to obtain a baculovirus recombinant, which is inoculated into a Bonbux Mori cocoon to obtain a Bonbux Mori cocoon emulsion. The resulting Bombux-Morri cocoon emulsion was produced by sonication in the same manner as in the Examples. Expression of the purified MERS coronavirus spike G protein can be confirmed by Western blotting in the same manner as in the Examples. From the amount of protein produced, an immune reaction can be predicted, and the effect in a neutralization test with the antibody produced thereby can be estimated. Further, the MERS coronavirus spike G protein can be fractionated in the same manner as in Example 1, and the component analysis of virus-like particles and the confirmation of virus-like particles by an electron microscope can be performed.

[Reference Example 4] Design of DNA for development of foot-and-mouth disease virus vaccine The present inventors have developed gene information for the expression of VP4-VP2-VP3-VP1-2A-3C fusion protein of foot-and-mouth disease virus in order to develop foot-and-mouth disease virus vaccine. To design. First, the amino acid sequences of VP4, Vp2, VP1, 2A, and 3C proteins in the amino acid sequence predicted from the nucleic acid sequence registered in Genbank under Accession No. HV940030 were linked from the N-terminal side to the C-terminal side. A FLAG tag sequence (SEQ ID NO: 5) was added to the amino acid sequence (SEQ ID NO: 31) to obtain an amino acid sequence of a foot-and-mouth disease virus serving as a basis for nucleic acid design (SEQ ID NO: 32). Based on the amino acid sequence of this foot-and-mouth disease virus VP4-VP2-VP3-VP1-2A-3C fusion protein, in the same manner as in Example 1, the foot-and-mouth disease virus codon-optimized VP4-VP2-VP3-VP1-2A-3C fusion protein A nucleic acid sequence was designed (SEQ ID NO: 33). FIG. 19 shows the correspondence between the nucleic acid sequence and amino acid sequence of the designed chimeric synthetic DNA. Based on the designed nucleic acid sequence of the foot-and-mouth disease virus codon-optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene, a full-length gene DNA was synthesized in the same manner as in the Examples, inserted into the pBm-8 vector, and pBM- 8fmdvP is created. A baculovirus recombinant is obtained using pBm-8fmdvP in the same manner as in Example 1, and inoculated into Bonbux Mori cocoons to obtain Bonbux Mori emulsion. The resulting Bombux-Morri cocoon emulsion was produced by sonication in the same manner as in the Examples. The expression of the purified foot-and-mouth disease virus VP4-VP2-VP3-VP1-2A-3C fusion protein was confirmed by Western blotting in the same manner as in the Examples, and the immune reaction could be predicted from the amount of protein produced. The effect in the neutralization test can be estimated. Moreover, the foot-and-mouth disease virus VP4-VP2-VP3-VP1-2A-3C fusion protein can be fractionated similarly to Example 1, and a virus-like particle | grain confirmation can be performed by the component analysis of an virus-like particle | grain and an electron microscope.

The present invention has the following features and remarkable effects:
(1) Since the gene sequence is designed using Codon Usage optimized for Bombux Mori cells and incorporated into the vector, the expression level of the recombinant viral membrane protein in Bombux Mori cells is the sequence derived from the viral gene. It is significantly higher than that using. (2) The obtained recombinant virus membrane protein constitutes a virus-like particle having a structure in which the surface of a spherical outer shell composed of a component containing a lipid derived from Bombux Mori is closely stuck as a spike. The particle size is 30 nm to 300 nm, which is the same size as an actual virus. Therefore, when the virus-like particle of the present invention is used as a vaccine, the immunogenicity (immunogenicity) is remarkably high, and it can be made comparable to the original virus.
(3) Since Bombyx Mori cells are used, the sugar chain structure is not complicated and the antigenic masking action by the sugar chain is very weak. Therefore, the immunogenicity (increased antibody titer) as a vaccine is remarkably high at 205 to 410 times that of a virus-derived purified HA protein grown in chicken eggs.
(4) The virus-like particle of the present invention substantially contains only a recombinant virus membrane protein prepared using artificially synthesized hybrid DNA and a lipid derived from Bombux moly, and does not contain other virus constituent proteins. Due to the high ratio of recombinant viral membrane proteins with high immunogenicity, the virus particles of the present invention can realize remarkably high immunogenicity when used as a vaccine. In this way, there is no other virus-like particle (artificial particle) having only recombinant virus membrane protein on the surface.
(5) A vaccine containing the virus-like particle of the present invention can induce a sufficiently high antibody titer not only by inoculation by injection but also by oral inoculation.
(6) By using artificially synthesized DNA, it is possible to produce a vaccine against a dangerous virus safely without handling the dangerous virus. P4 and P3 facilities are not required because dangerous viruses are not handled.
(7) For viral proteins used as vaccines, amino acid sequences with low pathogenicity can be designed based on bioinformatics. Thereby, a highly safe vaccine can be manufactured.
(8) The gene sequence is obtained from the amino acid sequence. In the amino acid sequence, for example, in the HA protein, except for the highly toxic site and the FLAG tag, it is exactly the same as the original amino acid sequence. Since its homology is only 77% (see, for example, FIG. 5), it can be easily distinguished from viral genes. Note that the use of a FLAG tag facilitates the confirmation and purification of expression.
(9) Since the recombinant virus membrane protein has a FLAG tag sequence at the C-terminal, even if the virus infects cells, it does not interact with the viral RNP or M1 protein, so it is safe Also excellent.
(10) When a vaccine is produced with eggs, a large amount of eggs is required, but a highly immunogenic vaccine as described above can be obtained by using Bombux Mori. The number of Bombux Moris can be reduced, and the cost can be reduced (about 1/10 of the method using chicken eggs).
(11) Compared with the case where vaccine is produced with a growing chicken egg, the time required for production can be greatly shortened when using Bombux Mori.
(12) Since the virus-like particle of the present invention has high immunogenicity, it is not necessary to add an adjuvant when used as a vaccine, and side effects such as anaphylactic shock and allergy are reduced, and the safety is excellent.

  According to the present invention, a highly immunogenic vaccine for preventing viral infection can be mass-produced at a low cost in a shorter time. In addition, by using Bonbux Mori, it is possible to solve the problem of side effects of egg allergy, and it is excellent in safety. Furthermore, the virus-like particle of the present invention exhibits sufficiently high immunogenicity even by oral inoculation. Therefore, the virus-like particle of the present invention, the production method thereof, and the vaccine containing the same are suitably used for vaccine production for the prevention of viral infections.

SEQ ID NO: 1: A / tufted duck / Fukushima / 16/2011 (H5N1) HA gene DNA sequence of avian influenza virus SEQ ID NO: 2: A / tufted duck / Fukushima / 16/2011 (H5N1) HA amino acid sequence of avian influenza virus No. 3: Modified HA amino acid sequence of A / tufted duck / Fukushima / 16/2011 (H5N1) avian influenza virus SEQ ID NO: 4: Codon-optimized HA gene of A / tufted duck / Fukushima / 16/2011 (H5N1) avian influenza virus DNA sequence SEQ ID NO: 5: FLAG tag amino acid sequence SEQ ID NO: 6: FLAG tag DNA sequence SEQ ID NO: 7: highly pathogenic amino acid sequence SEQ ID NO: 8: low pathogenic amino acid Acid sequence SEQ ID NO: 9: A / chicken / Sukabumi / 2008 (H5N1) avian influenza virus HA gene DNA SEQ ID NO: 10: A / chicken / Sukabumi / 2008 (H5N1) avian influenza virus HA amino acid sequence SEQ ID NO: 11: A / Chiken / Sukabumi / 2008 (H5N1) avian influenza virus modified HA amino acid sequence SEQ ID NO: 12: A / chicken / Sukabumi / 2008 (H5N1) avian influenza virus codon optimized HA gene DNA SEQ ID NO: 13: hepatitis C virus Core-E1-E2 fusion protein amino acid sequence SEQ ID NO: 14: Core-E1-E2 fusion protein amino acid sequence of hepatitis C virus (with FLAG tag) ) SEQ ID NO: 15: Codon-optimized Core-E1-E2 gene DNA sequence of hepatitis C virus SEQ ID NO: 16: HA amino acid sequence of avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) SEQ ID NO: 17: avian Modified HA amino acid sequence of influenza virus A / duck / Korea / A76 / 2010 (H7N7) SEQ ID NO: 18: Codon-optimized HA gene DNA sequence of avian influenza virus A / duck / Korea / A76 / 2010 (H7N7) SEQ ID NO: 19 Japanese encephalitis virus PreM-ME fusion protein amino acid sequence SEQ ID NO: 20: Japanese encephalitis virus PreM-ME fusion protein + FLAG tag amino acid sequence SEQ ID NO: 21: Japanese encephalitis virus codon optimized PreM-ME fusion tag Protein gene DNA sequence SEQ ID NO: 22: G protein amino acid sequence of rabies virus SEQ ID NO: 23: G protein of rabies virus + FLAG tag amino acid sequence SEQ ID NO: 24: Rabies virus codon optimized G protein gene DNA sequence SEQ ID NO: 25: West Nile virus PreM-ME fusion protein amino acid sequence SEQ ID NO: 26: West Nile virus PreM-ME fusion protein + FLAG tag amino acid sequence SEQ ID NO: 27: West Nile virus codon optimized PreM-ME fusion protein gene DNA sequence sequence Number 28: MERS coronavirus spike G protein amino acid sequence SEQ ID NO: 29: MERS coronavirus spike G protein + FLAG tag amino acid sequence SEQ ID NO: 30: MERS coronavirus codon optimization Pike G protein gene DNA SEQ ID NO: 31: VP4-VP2-VP3-VP1-2A-3C fusion protein amino acid sequence of foot-and-mouth disease virus SEQ ID NO: 32: VP4-VP2-VP3-VP1-2A-3C fusion protein + FLAG tag of foot-and-mouth disease virus Amino acid sequence SEQ ID NO: 33: Foot-and-mouth disease virus codon optimized VP4-VP2-VP3-VP1-2A-3C fusion protein gene DNA sequence

Claims (16)

  A virus-like particle comprising a recombinant viral membrane protein and a lipid derived from Bombus mori.   The virus-like particle according to claim 1, wherein spikes of the recombinant virus membrane protein are densely arranged on the surface of a hollow spherical outer shell composed of the lipid-containing component.   The virus-like particle according to claim 1 or 2, wherein the spherical outer shell has a diameter of 30 nm to 300 nm.   The virus-like particle according to claim 1, wherein the spike has a length of 5 nm to 30 nm.   The virus-like particle according to any one of claims 1 to 4, wherein a content weight ratio of the recombinant virus membrane protein and the lipid is 1: 1 to 1:10.   The virus-like particle according to any one of claims 1 to 5, wherein the recombinant virus membrane protein has an amino acid sequence encoded by a nucleic acid having a sequence codon-optimized for expression in Bombux Mori.   The virus-like particle according to any one of claims 1 to 6, wherein the recombinant virus membrane protein is a recombinant influenza virus HA protein.   The virus-like particle according to claim 7, wherein the influenza virus in the recombinant influenza virus HA protein is an influenza A virus.   The virus-like particle according to claim 8, wherein the influenza A virus is H5 or H7.   The virus-like particle according to any one of claims 1 to 6, wherein the recombinant virus membrane protein is a recombinant Japanese encephalitis virus protein.   The virus-like particle according to claim 6, wherein the amino acid sequence is selected from the group consisting of SEQ ID NOs: 3, 11, 14, 17, 20, 23, 26, 29, and 32.   The virus according to any one of claims 6 to 10, wherein the codon optimized DNA sequence is selected from the group consisting of SEQ ID NOs: 4, 12, 15, 18, 21, 24, 27, 30, and 33. Like particles.   The virus-like particle according to any one of claims 1 to 12, which is used for a vaccine.   A vaccine comprising the virus-like particle according to claim 1.   The vaccine according to claim 14, which is an oral vaccine. A step of obtaining a recombinant baculovirus by introducing a recombinant viral membrane protein gene having a sequence codon-optimized for expression in Bombux Mori into a baculovirus;
Inoculating the above-mentioned recombinant baculovirus into Bombyx Mori moth,
The step of rearing the pupae at 20-30 ° C. for 2-6 days,
A method for producing virus-like particles comprising the steps of producing an emulsion by crushing and emulsifying the pupae after breeding, and obtaining a fraction containing virus-like particles from the emulsion.