Construct for expressing e2 subunit of classical swine fever virus using baculovirus expression system, and use thereof

Abstract

To provide a technique for efficient production of a polypeptide usable to induce immunity against Classical Swine Fever Virus (CSFV) in pigs.SOLUTION: A polypeptide having a predetermined amino acid sequence is efficiently produced by using a construct including a polynucleotide encoding the amino acid sequence, the construct being for expressing the polypeptide by means of a baculovirus expression system.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a construct for expressing the E2 subunit of the porcine fever virus using a baculovirus expression system, and its use.

Background Art

[0002] Disease control by vaccines is considered an ideal countermeasure against infectious diseases because it is expected to achieve the maximum effect at the minimum cost.

[0003] Currently, the mainstream of vaccines is still inactivated vaccines or live attenuated vaccines that use the pathogen virus itself. On the other hand, subunit vaccines that produce specific virus-derived proteins as recombinant proteins and use them as vaccine antigens are expected to be safer vaccines because they do not contain the genome of the pathogenic virus. However, for many antigens that are difficult to mass-produce as recombinant proteins, manufacturing and development have not progressed.

[0004] For the production of recombinant proteins, various host systems such as bacteria, yeast, and cultured cells of mammals and insects are used, but each has its advantages and disadvantages. For example, Escherichia coli, which is commonly used in research, often has problems such as insolubilization of the produced protein and lack of the original function due to the absence of post-translational modification.

[0005] Among them, the baculovirus expression system is expected to be one of the high-expression systems for difficult-to-express proteins. As a baculovirus expression system, an expression system using insect cultured cells as a host is generally widely used worldwide. Insect cultured cells are excellent hosts as a recombinant protein expression system and are widely used because they can be mass-produced in a relatively simple aseptic environment.

[0006] Silkworms are completely domesticated insects that can be easily bred in large quantities, and their recombinant protein expression system is completed within an individual. Therefore, without large-scale equipment investment, scale-up can be achieved simply by increasing the number of times silkworms are bred, and there are many advantages such as not having to consider the breeding conditions for this. Regarding the production amount of recombinant proteins, especially for secreted proteins, the silkworm individual system produces far more than insect cultured cells.

[0007] Advantages of using the silkworm-baculovirus expression system in the recombinant protein expression system include that silkworms are domesticated insects that can be bred in large quantities, have high protein synthesis ability by using baculoviruses with high infectivity and proliferation ability, and can perform protein post-translational modifications similar to mammals. Furthermore, since the virus is inoculated into already grown individuals, there is no need to cause subsequent growth accompanied by cell division, and it is possible to produce proteins that show strong physiological activity that adversely affects cell proliferation. Silkworms grow from hatching to the last instar larvae in about 4 weeks, and during this period, their weight increases by about 10,000 times, so there is a possibility of synthesizing a large amount of protein.

[0008] Classical Swine Fever (CSF) is a disease of pigs and wild boars caused by infection with the CSF virus (CSFV). CSF is characterized by strong infectivity and high lethality and is designated as a livestock infectious disease in the Livestock Infectious Diseases Control Law. Therefore, in farms where CSF has occurred, it is stipulated that epidemic prevention measures against CSF should be taken for breeding pigs, etc.

[0009] To prevent the spread of the re-epidemic of classical swine fever in Japan in 2018, in the recommended vaccination areas, the live attenuated vaccine CSFV GPE- strain of CS F is used for pigs. Overseas, as a tool for classical swine fever eradication, in addition to live vaccines, a recombinant CSFV E2 subunit vaccine has been put into practical use.

[0010] Patent Document 1 and Patent Document 2 disclose a genetically recombinant CSFV E2 subunit vaccine. Patent Document 3 discloses a method for producing a vaccine containing a genetically recombinant CSFV E2 protein by a baculovirus expression system using insect cells. Non-Patent Document 1 reports the development of production technology for vaccines and diagnostic agents utilizing the silkworm-baculovirus expression system. Non-Patent Document 2 reports the development of a recombinant protein vaccine using silkworms, which is advantageous in terms of producing vaccine antigens (especially virus-like particles) that are difficult to mass-produce.

[0011] In view of the threat of CSF that causes significant economic losses to fields relying on commercial pig farming and its production products, the development of vaccines and vaccination programs that can effectively prevent CSFV infection and the occurrence of CSF is eagerly desired as diverse options for epidemic prevention in Japan.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0014] An object of the present invention is to provide a technique for efficiently producing a polypeptide that can be used to induce immunity against CSFV in pigs.

Means for Solving the Problems

[0015] The present inventors constructed a construct containing a polynucleotide encoding the CSFV E2 subunit, expressed the subunit using a baculovirus expression system, and inoculated pigs with a vaccine containing the same, and found that immunity against CSFV can be successfully induced in the pigs. Based on these findings, the present inventors completed the present invention.

[0016] That is, the present invention can be exemplified as follows. [1] A construct containing a polynucleotide encoding an amino acid sequence of a polypeptide consisting of the amino acid sequence of any one of the following (1), (2), or (3) for expression using a baculovirus expression system: (1) The amino acid sequence of the E2 subunit found in a CSFV epidemic field strain (SEQ ID NO: 2); (2) An amino acid sequence including substitution, deletion, insertion, and / or addition of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and having a function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having a function of producing an antibody having neutralizing activity against CSFV. [2] The construct contains a polynucleotide encoding a secretory signal peptide, The polypeptide is the one with a secretory signal peptide added to the polypeptide, The construct according to [1]. [3] The construct according to [2], wherein the secretion signal peptide is the secretion signal peptide of the E2 subunit found in the CSFV epidemic field strain or the secretion signal peptide of the silkworm 30K protein. [4] The construct contains a polynucleotide encoding a soluble tag peptide, The polypeptide is one to which a soluble tag peptide is added to the polypeptide, The construct according to any one of [1] to [3]. [5] The construct according to [4], wherein the soluble tag peptide is ZZ or SUMO. [6] The construct contains a polynucleotide encoding a tag peptide for protein purification, The polypeptide is one to which a tag peptide for protein purification is added to the polypeptide, The construct according to any one of [1] to [5]. [7] The construct according to [6], wherein the tag peptide for protein purification is a His tag or a Strep tag. [8] A recombinant baculovirus into which the construct according to any one of [1] to [7] is introduced.

[0017] [9] A method for producing a polypeptide, The polypeptide is a polypeptide consisting of the amino acid sequence of (1), (2), or (3) below, The method includes a recovery step of recovering the polypeptide expressed in a baculovirus-susceptible insect infected with the recombinant baculovirus according to [8] from the insect. Production method: (1) The amino acid sequence of the E2 subunit found in the porcine fever virus (CSFV) epidemic field strain (SEQ ID NO: 2); (2) An amino acid sequence that contains substitution, deletion, insertion, and / or addition of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and has the function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence that has 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and has the function of producing an antibody having neutralizing activity against CSFV.

[10] The production method according to [9], wherein the recovery step is a step of recovering the polypeptide from the serum of the insect.

[11] The production method according to [9] or

[10] , wherein the insect is a silkworm.

[12] A subunit vaccine against classical swine fever virus (CSFV), The subunit vaccine comprising a polypeptide consisting of the amino acid sequence of any one of the following (1), (2), or (3): (1) The amino acid sequence of the E2 subunit found in a classical swine fever virus (CSFV) epidemic field strain (SEQ ID NO: 2); (2) An amino acid sequence that contains substitution, deletion, insertion, and / or addition of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and has the function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence that has 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and has the function of producing an antibody having neutralizing activity against CSFV.

Advantages of the Invention

[0018] The present invention can provide a technique for efficiently producing a polypeptide that can be used to induce immunity against CSFV in pigs.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Mode for Carrying Out the Invention

[0020] <1>Vaccine The vaccine described in this specification is a vaccine against Classical Swine Fever (CSF).

[0021] "Classical Swine Fever (CSF)" means a disease of pigs caused by infection with Classical Swine Fever Virus (CSFV).

[0022] The vaccine described in this specification is a CSFV E2 subunit vaccine. "CSFV E2 subunit vaccine" means a vaccine containing the E2 subunit of CSFV as an active ingredient. This vaccine is hereinafter also simply referred to as "subunit vaccine". The terms "CSFV E2 subunit vaccine", "subunit vaccine", "CSFV E2 subunit vaccine against CSFV", and "subunit vaccine against CSFV" may be used interchangeably with each other.

[0023] "Subunit vaccine" means a subunit vaccine capable of inducing immunity against CSFV in pigs. Specifically, "subunit vaccine" may mean a subunit vaccine capable of inducing immunity against CSFV in a pig by inoculating the pig. That is, by using a subunit vaccine, specifically, by inoculating a pig with a subunit vaccine, immunity against CSFV can be induced in the pig, that is, an effect of inducing immunity against CSFV in the pig can be obtained. This effect is also referred to as the "immunity induction effect".

[0024] The "subunit vaccine" may be the "CSF booster vaccine". The "CSF booster vaccine" means a vaccine capable of enhancing immunity against CSFV in pigs that already have immunity against CSFV. When the "subunit vaccine" is the "CSF booster vaccine", "inducing immunity against CSFV in pigs" may be "enhancing immunity against CSFV in pigs". That is, "inducing immunity against CSFV in pigs" may include "enhancing immunity against CSFV in pigs". Specifically, the "CSF booster vaccine" may mean a vaccine capable of enhancing immunity against CSFV in pigs by inoculating pigs that already have immunity against CSFV. That is, by using the booster vaccine, specifically, by inoculating a pig that already has immunity against CSFV with the booster vaccine, the immunity against CSFV in the pig can be enhanced, that is, the effect of enhancing the immunity against CSFV in the pig can be obtained. This effect is also referred to as the "booster (effect)". When the "subunit vaccine" is the "CSF booster vaccine", the "immune induction effect" may be the "booster (effect)". That is, the "immune induction effect" may include the "booster (effect)".

[0025] The terms "immunity against CSFV" and "immunity against CSF" may be used interchangeably. The terms "antibody against CSFV" and "antibody against CSF" may be used interchangeably.

[0026] The E2 subunit of CSFV is hereinafter also simply referred to as the "E2 subunit". That is, the E2 subunit functions as an active ingredient (specifically, the active ingredient of a subunit vaccine). "The E2 subunit functions as an active ingredient" or "the E2 subunit functions as the active ingredient of a subunit vaccine" means that an immune induction effect is obtained depending on the presence of the E2 subunit. Also, "the E2 subunit functions as an active ingredient" or "the E2 subunit functions as the active ingredient of a subunit vaccine" may mean having a function of producing an antibody having neutralizing activity against CSFV. Details of the E2 subunit will be described later.

[0027] The subunit vaccine may consist of the E2 subunit, or may contain components other than the E2 subunit in addition to the E2 subunit. Components other than the E2 subunit that may be contained in the subunit vaccine are also referred to as "additional components". The additional components may or may not contribute to the immune induction effect.

[0028] Examples of additional components include components that can be normally contained in vaccines such as vaccines for pigs. Such components include adjuvants, preservatives, and solvents.

[0029] The subunit vaccine can be produced, for example, by appropriately mixing its raw materials (that is, the E2 subunit and optionally additional components).

[0030] The content and content ratio of each component in the subunit vaccine (i.e., the E2 subunit and optionally additional components) are not particularly limited as long as an immune induction effect can be obtained by using the subunit vaccine. The content and content ratio of each component in the subunit vaccine (i.e., the E2 subunit and optionally additional components) can be appropriately set according to various conditions such as the type of each component and the usage mode of the subunit vaccine. For example, the content of the E2 subunit in the subunit vaccine may be an amount that can obtain an immune induction effect when the subunit vaccine is used in the manner described in the method described in this specification below.

[0031] The form of the subunit vaccine is not particularly limited. The subunit vaccine may be in any form such as a liquid or a powder. In particular, the subunit vaccine may be a liquid. The subunit vaccine may be produced and provided, for example, in a form that can be used as it is, or in a form that requires preparation at the time of use. The subunit vaccine may be produced and provided, for example, in a form that is diluted with a solvent at the time of use. Examples of the form that is diluted with a solvent at the time of use include concentrated forms such as dried products and concentrated solutions.

[0032] The subunit vaccine can be used, for example, in the manner described in the method described in this specification below. That is, the subunit vaccine may be, for example, for use in the method described in this specification below.

[0033] Specifically, the subunit vaccine may be for inoculating pigs that have no vaccination history against CSFV live vaccine (also referred to as "CSFV live vaccine" or "live vaccine of CSFV"), or may be for inoculating pigs that have been inoculated with CSFV live vaccine.

[0034] When the subunit vaccine is a booster vaccine, specifically, the booster vaccine may be for inoculating pigs that have been inoculated with CSFV live vaccine.

[0035] The subunit vaccine may specifically be for inducing immunity against CSFV in pigs. More specifically, the subunit vaccine may be for inoculating pigs without a history of inoculation with live CSFV vaccine in order to induce immunity against CSFV in pigs, or may also be for inoculating pigs that have been inoculated with live CSFV vaccine.

[0036] When the subunit vaccine is a booster vaccine, the booster vaccine may specifically be for enhancing immunity against CSFV in pigs. More specifically, the booster vaccine may be for inoculating pigs that have been inoculated with live CSFV vaccine in order to enhance immunity against CSFV in pigs.

[0037] The subunit vaccine may specifically be for preventing infection with CSFV in pigs, for preventing the occurrence of CSF in pigs, and / or for reducing symptoms during the occurrence of CSF in pigs. More specifically, the subunit vaccine may be for inoculating pigs without a history of inoculation with live CSFV vaccine in order to prevent infection with CSFV in pigs, for preventing the occurrence of CSF in pigs, and / or for reducing symptoms during the occurrence of CSF in pigs, or may also be for inoculating pigs that have been inoculated with live CSFV vaccine.

[0038] When the subunit vaccine is a booster vaccine, the booster vaccine may specifically be for preventing infection with CSFV in pigs, for preventing the occurrence of CSF in pigs, and / or for reducing symptoms during the occurrence of CSF in pigs. More specifically, the booster vaccine may be for inoculating pigs that have been inoculated with live CSFV vaccine in order to prevent infection with CSFV in pigs, for preventing the occurrence of CSF in pigs, and / or for reducing symptoms during the occurrence of CSF in pigs.

[0039] The subunit vaccine may specifically be for conferring immunity against CSFV to piglets. More specifically, the subunit vaccine may be for inoculating sows that have no vaccination history with live CSFV vaccine and that nurse the piglets with colostrum, or may be for inoculating sows that have been vaccinated with live CSFV vaccine and that nurse the piglets with colostrum, in order to confer immunity against CSFV to the piglets.

[0040] When the subunit vaccine is a booster vaccine, the booster vaccine may specifically be for conferring immunity against CSFV to piglets. More specifically, the booster vaccine may be for inoculating sows that have been vaccinated with live CSFV vaccine and that nurse the piglets with colostrum, in order to confer immunity against CSFV to the piglets.

[0041] The subunit vaccine may specifically be for preventing CSFV infection in piglets, for preventing the occurrence of CSF in piglets, and / or for alleviating the symptoms when CSF occurs in piglets. More specifically, the subunit vaccine may be for preventing CSFV infection in piglets, for preventing the occurrence of CSF in piglets, and / or for alleviating the symptoms when CSF occurs in piglets, in order to inoculate sows that have no vaccination history with live CSFV vaccine and that nurse the piglets with colostrum, or may be for inoculating sows that have been vaccinated with live CSFV vaccine and that nurse the piglets with colostrum.

[0042] ​When the subunit vaccine is a booster vaccine, the booster vaccine may specifically be for preventing the infection of CSFV in piglets, for preventing the occurrence of CSF in piglets, and / or for reducing the symptoms during the occurrence of CSF in piglets. More specifically, the booster vaccine may be for inoculating a sow that has been inoculated with a live CSFV vaccine and that nurses the piglet with colostrum for preventing the infection of CSFV in the piglet, for preventing the occurrence of CSF in the piglet, and / or for reducing the symptoms during the occurrence of CSF in the piglet.

[0043] <2>E2 subunit of CSFV The "E2 subunit of CSFV" means a polypeptide containing the amino acid sequence of the E2 subunit found in any strain of CSFV or its variant sequence. The variant sequence may or may not be found in any strain of CSFV. The said sequence contained in the E2 subunit (that is, the amino acid sequence of the E2 subunit found in any strain of CSFV or its variant sequence) is also referred to as the "E2 subunit sequence".

[0044] The "E2 subunit of a certain CSFV strain" means a polypeptide containing the E2 subunit sequence derived from the certain CSFV strain (that is, the amino acid sequence of the E2 subunit found in the certain CSFV strain or its variant sequence). The variant sequence may or may not be found in the certain CSFV strain.

[0045] The E2 subunit may be encoded by the E2 subunit gene. That is, the "E2 subunit gene" means a polynucleotide encoding the E2 subunit. The nucleotide sequence of the E2 subunit gene is not particularly limited as long as it encodes the E2 subunit. The E2 subunit gene may be, for example, a polynucleotide containing a nucleotide sequence encoding the E2 subunit found in any strain of CSFV or a variant sequence thereof. The variant sequence may or may not be found in any strain of CSFV.

[0046] The CSFV strain from which the E2 subunit sequence or the nucleotide sequence encoding it is derived is not particularly limited as long as the E2 subunit functions as an active ingredient. Examples of the CSFV strain from which the E2 subunit sequence or the nucleotide sequence encoding it is derived include CSFV wild strains and CSFV attenuated strains. In particular, examples of the CSFV strain from which the E2 subunit sequence or the nucleotide sequence encoding it is derived include CSFV wild strains. Examples of CSFV wild strains include CSFV field epidemic strains. That is, the E2 subunit may be, for example, the E2 subunit of a CSFV field epidemic strain, and specifically, a polypeptide containing an E2 subunit sequence derived from a CSFV field epidemic strain (that is, an amino acid sequence of the E2 subunit found in a CSFV field epidemic strain or a variant sequence thereof).

[0047] The nucleotide sequence encoding the E2 subunit found in a CSFV field epidemic strain is shown as SEQ ID NO: 1, and the amino acid sequence of the same E2 subunit is shown as SEQ ID NO: 2. That is, the E2 subunit gene may be, for example, a polynucleotide containing the nucleotide sequence shown in SEQ ID NO: 1 or a variant sequence thereof. Also, the E2 subunit may be, for example, a polypeptide containing the amino acid sequence shown in SEQ ID NO: 2 or a variant sequence thereof.

[0048] The variant sequence of the nucleotide sequence for the E2 subunit gene and the variant sequence of the amino acid sequence for the E2 subunit may each be a naturally occurring variant or an artificially modified variant. The variant sequence of the nucleotide sequence for the E2 subunit gene is not particularly limited as long as the polynucleotide containing the variant sequence encodes the E2 subunit. The variant sequence of the amino acid sequence for the E2 subunit is not particularly limited as long as the polypeptide containing the variant sequence functions as an active ingredient (specifically, the active ingredient of a subunit vaccine).

[0049] That the polypeptide functions as an active ingredient (specifically, the active ingredient of a subunit vaccine) can be confirmed, for example, by inoculating the polypeptide into pigs (for example, pigs without a vaccination history of live CSF vaccine or pigs that have been inoculated with live CSF vaccine) and confirming an increase in the antibody titer against CSFV. The inoculation of the polypeptide into pigs and the measurement of the antibody titer can be carried out, for example, in the same procedure as the inoculation of the subunit vaccine into pigs and the measurement of the antibody titer in the method described in this specification, which will be described later.

[0050] That the polypeptide functions as an active ingredient (specifically, the active ingredient of a subunit vaccine) can be confirmed, for example, by confirming that an antigen-antibody reaction occurs between the polypeptide and an anti-E2 subunit antibody. The antigen-antibody reaction can be measured, for example, by a conventional method.

[0051] Examples of the variant sequence of the amino acid sequence for the E2 subunit include amino acid sequences in which one or several amino acids are substituted, deleted, inserted, and / or added at one or several positions in the above amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2). The above "one or several" may be, for example, 1 to 65, 1 to 60, 1 to 55, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, or 1.

[0052] As a variant sequence of the amino acid sequence for the E2 subunit, there are also amino acid sequences having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity with the entire amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 2).

[0053] As a variant sequence of the nucleotide sequence for the E2 subunit gene, there is a nucleotide sequence that hybridizes with the complementary sequence of the above nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 1) under stringent conditions. "Stringent conditions" may mean conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. As stringent conditions, there are conditions for washing in ordinary Southern hybridization, such as 60 ° C, 1 × SSC, 0.1% SDS, preferably 60 ° C, 0.1 × SSC, 0.1% SDS, more preferably 68 ° C, 0.1 × SSC, 0.1% SDS, and washing once, preferably 2 to 3 times, at the corresponding salt concentration and temperature.

[0054] As a variant sequence of the nucleotide sequence for the E2 subunit gene, there are also nucleotide sequences having 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity with the entire nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 1).

[0055] As a variant sequence of the nucleotide sequence for the E2 subunit gene, there is also a nucleotide sequence in which any one or more codons in the above nucleotide sequence (for example, the nucleotide sequence shown in SEQ ID NO: 1) are replaced with equivalent codons (that is, a variant sequence of the above nucleotide sequence due to codon degeneracy). The variant sequence of the nucleotide sequence may be modified, for example, to have optimal codons according to the codon usage frequency of the host used for heterologous expression.

[0056] The identity between amino acid sequences and the identity between nucleotide sequences can both be calculated, for example, by conventional methods. Specifically, the identity between amino acid sequences can be calculated, for example, by blastp. Specifically, the identity between nucleotide sequences can be calculated, for example, by blastn.

[0057] The E2 subunit may be a polypeptide consisting of an E2 subunit sequence, or may be a polypeptide consisting of an amino acid sequence with one or more amino acid residues added to the N-terminus and / or C-terminus of the E2 subunit sequence. The amino acid residues that can be added to the N-terminus and / or C-terminus of the E2 subunit sequence are also referred to as "added sequences". The added sequences are not particularly limited as long as the E2 subunit functions as an active ingredient (specifically, the active ingredient of a subunit vaccine). The added sequences can be independently selected for the N-terminus and C-terminus of the E2 subunit sequence, respectively.

[0058] Examples of added sequences include signal peptides and peptide tags. The term "peptide tag" may be used as the term "tag peptide". The term "tag peptide" may be used to indicate the peptide constituting the peptide tag.

[0059] As the signal peptide, for example, one that functions in the expression host of the E2 subunit may be selected. "The signal peptide functions in the expression host of the E2 subunit" may mean that the signal peptide contributes to the transport of the E2 subunit to the endoplasmic reticulum or extracellular space in the expression host. Examples of signal peptides include secretory signal peptides, etc. Specifically, for example, the secretory signal peptide of the E2 subunit found in CSFV field epidemic strains, the secretory signal peptide of the silkworm 30K protein, etc. can be mentioned.

[0060] Examples of peptide tags include His tag, FLAG tag, GST tag, Myc tag, ZZ tag, SUMO tag, Strep tag, MBP (maltose binding protein), CBP (cellulose binding protein), TRX (Thioredoxin), GFP (green fluorescent protein), HRP (horseradish peroxidase), ALP (Alkaline Phosphatase), and the Fc region of an antibody. The peptide tag may be used, for example, for the detection or purification of the expressed E2 subunit. Examples of peptide tags include solubilizing peptide tags (specifically, peptide tags that may improve solubility), and specifically, for example, ZZ tag, SUMO tag, etc. Also included are peptide tags for protein purification, and specifically, for example, His tag, Strep tag, etc.

[0061] The additional sequence may, for example, be present during the expression of the E2 subunit but not in the ultimately obtained E2 subunit. For example, the signal peptide may be cleaved after the expression of the E2 subunit and thus may not be present in the ultimately obtained E2 subunit.

[0062] <3>Construct The E2 subunit gene may be included in the construct. A construct containing the E2 subunit gene may be a vector or nucleic acid fragment containing the E2 subunit gene. That is, it may be a vector or nucleic acid fragment containing a polynucleotide encoding the E2 subunit.

[0063] A construct containing an E2 subunit gene may be, for example, a construct containing a polynucleotide encoding an E2 subunit found in any strain of CSFV. For example, it may be a construct containing a polynucleotide encoding a polypeptide containing the amino acid sequence corresponding to amino acid numbers 1 to 331 of the amino acid sequence of the E2 subunit found in a CSFV field epidemic strain (the amino acid sequence shown in SEQ ID NO: 2) or a variant sequence thereof. The polypeptide may be a polypeptide consisting of the amino acid sequence corresponding to amino acid numbers 1 to 331 of the amino acid sequence of the E2 subunit found in a CSFV field epidemic strain ( the amino acid sequence shown in SEQ ID NO: 2) or a variant sequence thereof.

[0064] The amino acid sequence corresponding to amino acid numbers 1 to 331 of the amino acid sequence of the E2 subunit found in the CSFV field epidemic strain (the amino acid sequence shown in SEQ ID NO: 2) or a variant sequence thereof may be, for example, the amino acid sequence of any of the following (1), (2), or (3). That is, the construct may be a construct containing a polynucleotide encoding the amino acid sequence for expressing a polypeptide containing the amino acid sequence of any of the following (1), (2), or (3). The polypeptide may be a polypeptide consisting of the amino acid sequence of any of the following (1), (2), or (3). (1) The amino acid sequence of the E2 subunit found in a porcine fever virus (CSFV) field epidemic strain (SEQ ID NO: 2); (2) An amino acid sequence containing substitutions, deletions, insertions, and / or additions of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and having the function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having the function of producing an antibody having neutralizing activity against CSFV.

[0065] Constructs containing the E2 subunit gene may contain a polynucleotide encoding amino acid residues (i.e., additional sequences) that can be added to the N-terminus and / or C-terminus of the E2 subunit sequence, as exemplified previously.

[0066] <4>Method for producing the E2 subunit As the E2 subunit, those appropriately produced and obtained may be used.

[0067] The method for producing the E2 subunit is not particularly limited. The E2 subunit can be produced, for example, by expressing the E2 subunit gene in a host having the E2 subunit gene. The host may have the E2 subunit gene in the form of a construct containing the E2 subunit gene. Also, the E2 subunit can be produced, for example, by expressing the E2 subunit gene in a cell-free protein synthesis system. Further, the E2 subunit can be produced, for example, by separating and purifying it from CSFV.

[0068] Hereinafter, the production of the E2 subunit using a host having the E2 subunit gene will be exemplified. The E2 subunit produced using a host having the E2 subunit gene is also referred to as a "recombinant E2 subunit".

[0069] The host is not particularly limited as long as it can express the E2 subunit that functions as an active ingredient (specifically, the active ingredient of the subunit vaccine). Examples of the host include microorganisms, insect cells, plant cells, and animal cells. The cells may form an aggregate such as a tissue or an organism, for example.

[0070] Examples of microorganisms include bacteria and yeasts. Examples of bacteria include bacteria belonging to the family Enterobacteriaceae, coryneform bacteria, and bacteria belonging to the genus Bacillus. Examples of bacteria belonging to the family Enterobacteriaceae include bacteria belonging to the genus Escherichia such as Escherichia coli. Examples of coryneform bacteria include bacteria belonging to the genus Corynebacterium such as Corynebacterium glutamicum. An example of a bacterium belonging to the genus Bacillus is Bacillus subtilis.

[0071] Examples of insects include insects belonging to the genus Autographa such as Autographa californica, insects belonging to the genus Trichoplusia such as Trichoplusia ni, insects belonging to the genus Bombyx such as Bombyx mori, insects belonging to the genus Orgyia such as Orgyia pseudotsugata, insects belonging to the genus Lymantria such as Lymantria dispar, and insects belonging to the genus Spodoptera such as Spodoptera exigua and Spodoptera frugiperda.

[0072] Examples of plants include those of the Solanaceae family and the Brassicaceae family. Examples of Solanaceae plants include those of the Nicotiana genus and the Solanum genus. Examples of Nicotiana plants include Nicotiana benthamiana and Nicotiana tabacum. Examples of Solanum plants include Solanum lycopersicum, Solanum melongena, and Solanum tuberosum. Examples of Brassicaceae plants include those of the Arabidopsis genus. Examples of Arabidopsis plants include Arabidopsis thaliana.

[0073] Examples of animals include mammals. Examples of mammals include rodents and primates. Examples of rodents include hamsters, mice, rats, and guinea pigs. Examples of hamsters include Chinese hamsters. Examples of primates include humans, monkeys, and chimpanzees. Examples of monkeys include African green monkeys.

[0074] A host having the E2 subunit gene can be obtained, for example, by introducing the E2 subunit gene into the host. Introducing the E2 subunit gene into the host may be by introducing it into the host in the form of a construct containing the E2 subunit gene.

[0075] The E2 subunit gene can be obtained, for example, by cloning from CSFV or by chemical synthesis. The obtained E2 subunit gene can be used as it is or after appropriate modification. Modification of the gene can be carried out by known methods. For example, the target mutation can be introduced into the target site of DNA by site-directed mutagenesis. Examples of site-directed mutagenesis methods include the method using PCR (Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)) and the method using phage (Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)).

[0076] The method for introducing the E2 subunit gene into the host is not particularly limited. The E2 subunit gene only needs to be retained in the host so that it can be expressed. Specifically, in the host, the E2 subunit gene only needs to be retained so that it can be expressed under the control of a promoter that functions in the host. In the host, the E2 subunit gene may be present, for example, on a vector that replicates autonomously outside the genome or may be introduced into the genome.

[0077] The promoter for expressing the E2 subunit gene is not particularly limited as long as it functions in the host. The "promoter that functions in the host" may mean a promoter having promoter activity in the host. The promoter may be a promoter derived from the host or a promoter derived from a heterologous source. The promoter may be the native promoter of the E2 subunit gene or the promoter of another gene. For example, promoters that function in bacteria of the family Enterobacteriaceae such as E. coli include the T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, msrA promoter, Pm1 promoter, PR promoter, and PL promoter. Also, for example, promoters that function in coryneform bacteria such as C. glutamicum include the lac promoter, tac promoter, trc promoter, F1 promoter, P54-6 promoter (Appl. Microbiol. Biotechnol., 53, 674-679(2000)), EF-Tu promoter (Journal of Biotechnology 104 (2003) 311-323, Appl. Environ. Microbiol. 2005 Dec;71(12):8587-96.), SOD promoter, and promoters of genes such as pta, aceA, aceB, adh, amyE, and cspB. Also, for example, promoters that function in insect cells include promoters derived from baculoviruses such as the polyhedrin promoter. Examples of baculoviruses include AcMNPV, BmNPV, OpMNPV, LdMNPV, and SeMNPV. Therefore, the construct containing the E2 subunit gene may be introduced into a baculovirus. The baculovirus into which the construct containing the E2 subunit gene has been introduced is also referred to as a "recombinant baculovirus". Also, for example, promoters that function in plant cells include plant-derived promoters and plant virus-derived promoters.Examples of plant-derived promoters include the ubiquitin promoter. Examples of plant viruses include Cauliflower mosaic virus (CaMV), Commelina yellow mottle virus (CoYMV), Rice tungro bacilliform virus (RTBV), Sugarcane bacilliform virus (SCBV), Soybean chlorotic mottle virus (SbCMV), Figwort mosaic virus (FMV), Carnation etched ring virus (CERV), Peanut chlorotic streak virus (PCSV), Strawberry vein banding virus (SVBV), Cacao swollen shoot virus (CSSV), and Cassava vein mosaic virus (CsVMV). Examples of promoters that function in animal cells include the SV40 promoter, EF1a promoter, RSV promoter, CMV promoter, and SRalpha promoter.

[0078] The E2 subunit gene can be introduced into a host, for example, by introducing a vector containing the gene into the host. Introducing a vector containing the gene into the host may be to introduce it into the host in the form of a construct containing the E2 subunit gene. A vector containing the E2 subunit gene is also referred to as an "expression vector for the E2 subunit gene". The expression vector for the E2 subunit gene can be constructed, for example, by ligating a DNA fragment containing the E2 subunit gene to a vector. By transforming a host with the expression vector for the E2 subunit gene, the gene can be introduced into the host. The vector may or may not be capable of autonomous replication in the host. The vector may be equipped with a marker such as a drug resistance gene. The vector may be equipped with an expression regulatory sequence such as a promoter for expressing the E2 subunit gene. The vector can be appropriately selected according to various conditions such as the type of the host. Examples of the vector include plasmid vectors and viral vectors. For example, vectors capable of autonomous replication in bacteria of the family Enterobacteriaceae such as E. coli include pUC vectors such as pUC19 and pUC18, pHSG vectors such as pHSG299, pHSG399, and pHSG398, pET vectors, pTrc99A, and pBR322. Also, for example, vectors capable of autonomous replication in coryneform bacteria such as C. glutamicum include pHM1519, pAM330, pVK4, pVK7, pVC7, and pVS7. Also, for example, vectors available for use in insect cells include plasmid vectors such as pIZ / V5-His and viral vectors derived from baculoviruses as exemplified above. Also, for example, vectors available for use in plant cells include plasmid vectors such as pRI vectors and pCambia vectors and viral vectors derived from plant viruses as exemplified above. Also, for example, vectors available for use in animal cells include plasmid vectors such as pcDNA vectors, pBApo-CMV vectors, and pCI-neo (Promega), and viral vectors such as retroviral vectors and adenoviral vectors.Depending on the type and composition of the vector, the expression vector of the E2 subunit gene may be integrated into the host genome, may replicate autonomously outside the host genome, or may be temporarily retained outside the host genome.

[0079] In addition, the E2 subunit gene can be introduced into a host, for example, by introducing a nucleic acid fragment containing the gene into the host. Examples of such nucleic acid fragments include linear DNA and linear RNA.

[0080] The method for introducing nucleic acids such as vectors and nucleic acid fragments into a host can be appropriately selected according to various conditions such as the type of the host. Examples of the method for introducing nucleic acids into a host include the DEAE dextran method, the calcium phosphate method, the lipofection method, the electroporation method, and the microinjection method. When the vector is a viral vector, the vector (virus) can be introduced into the host by infecting the host with the vector.

[0081] The expressed E2 subunit may be recovered, for example, as a solution containing the E2 subunit and used as an active ingredient of a subunit vaccine. Examples of the solution containing the E2 subunit include a culture solution of a host, a culture supernatant of a host, and a cell disruption extract of a host. When the host is an insect, body fluid (for example, serum, etc.) may be mentioned. Examples of the insect include the insects already exemplified. For example, it may be a silkworm. Taking as an example the case where a construct containing the E2 subunit gene is introduced into a baculovirus (that is, explaining with a recombinant baculovirus as an example), the E2 subunit expressed in the body of a baculovirus-sensitive insect infected with the recombinant baculovirus may be recovered from the insect. The E2 subunit may be recovered from the serum of the insect. Specifically, a polypeptide containing the amino acid sequence of any of the following (1), (2), or (3) expressed in the body of a baculovirus-sensitive insect infected with the recombinant baculovirus may be recovered from the insect. The polypeptide may be recovered from the serum of the insect. The polypeptide may be a polypeptide consisting of the amino acid sequence of any of the following (1), (2), or (3). (1) The amino acid sequence of the E2 subunit found in a classical swine fever virus (CSFV) epidemic field strain (SEQ ID NO: 2); (2) An amino acid sequence that includes substitution, deletion, insertion, and / or addition of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and has a function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having a function of producing an antibody having neutralizing activity against CSFV.

[0082] In addition, the expressed E2 subunit may be purified to a desired degree and used as an active ingredient of a subunit vaccine, for example.

[0083] <5> Method A first aspect of the method described in this specification is a method for inducing immunity against CSFV in pigs. Hereinafter, this method (i.e., the first aspect of the method described in this specification) is also referred to as the "first method".

[0084] The first method includes inoculating a pig with a subunit vaccine.

[0085] The pig inoculated with the subunit vaccine may be a pig without a history of inoculation with a live CSFV vaccine or a pig that has been inoculated with a live CSFV vaccine. The pig inoculated with the subunit vaccine is not particularly limited other than having no history of inoculation with a live CSFV vaccine or having been inoculated with a live CSFV vaccine. That is, as the pig inoculated with the subunit vaccine, any pig that desires (needs) to induce immunity against CSVF and has no history of inoculation with a live CSFV vaccine or has been inoculated with a live CSFV vaccine may be selected. A pig without a history of inoculation with a live CSFV vaccine may or may not already have immunity against CSFV. A pig that has been inoculated with a live CSFV vaccine may already have immunity against CSFV.

[0086] When the subunit vaccine is a booster vaccine, the pig inoculated with the booster vaccine may be a pig that has been inoculated with a live CSFV vaccine. At this time, the pig inoculated with the booster vaccine is not particularly limited other than having been inoculated with a live CSFV vaccine. That is, as the pig inoculated with the booster vaccine, any pig that desires (needs) to enhance immunity against CSVF and has been inoculated with a live CSFV vaccine may be selected. When the subunit vaccine is a booster vaccine, "desires (needs) to induce immunity against CSVF" may be "desires (needs) to enhance immunity against CSVF". A pig that has been inoculated with a live CSFV vaccine may already have immunity against CSFV.

[0087] Examples of the live CSFV vaccine include a CSFV attenuated strain. Examples of the CSFV attenuated strain include the LPC strain, C strain, GPE- strain, LOM strain, CP7_E2alf strain, Flc-LOM-BErns Examples of strains include CSFV attenuated strains, and particularly, GPE- strains.

[0088] The number of inoculations of live vaccine in pigs already inoculated with CSFV live vaccine is not particularly limited. The number of inoculations of live vaccine may be, for example, 1 time, or may be 2 times or more. The number of inoculations of live vaccine may be, for example, 5 times or less, 4 times or less, 3 times or less, or 2 times or less. The number of inoculations of live vaccine may be, for example, within a range defined by any combination of the above-exemplified ranges. The number of inoculations of live vaccine may particularly be 1 time or 2 times.

[0089] When the number of inoculations of live vaccine in pigs already inoculated with CSFV live vaccine is 2 times or more, the implementation modes of each inoculation (for example, conditions related to inoculation such as the type of live vaccine, inoculation route, inoculation amount, etc.) may or may not be the same. When the number of inoculations of live vaccine in pigs already inoculated with CSFV live vaccine is 2 times or more, the implementation modes of each inoculation may particularly be the same.

[0090] The first method may or may not include inoculating pigs with CSFV live vaccine before inoculating with subunit vaccine.

[0091] When the first method includes inoculating pigs with CSFV live vaccine before inoculating with subunit vaccine, the pigs after inoculation with the CSFV live vaccine may be regarded as the pigs to be inoculated with the subunit vaccine in the first method. That is, specifically, the first method may include inoculating pigs with CSFV live vaccine and inoculating the pigs after inoculation with the CSFV live vaccine with subunit vaccine. The pigs inoculated with CSFV live vaccine may be pigs without a CSFV live vaccine inoculation history, or may be pigs already inoculated with CSFV live vaccine 1 time or more.

[0092] The pigs to be inoculated with the subunit vaccine may be female or male. The pigs to be inoculated with the subunit vaccine may be, for example, breeding pigs. "Breeding pigs" means pigs used for breeding, and specifically, may collectively refer to female and male breeding pigs. The breeding pigs may be multiparous or nulliparous. The purpose of breeding of the breeding pigs is not particularly limited. The purpose of breeding of the breeding pigs may be, for example, for meat use. The pigs to be inoculated with the subunit vaccine (for example, breeding pigs) may specifically be breeding pigs from the second generation and later, or may not be. The pigs to be inoculated with the subunit vaccine (for example, breeding pigs) may particularly be female pigs.

[0093] "The breeding pigs of the second generation and later" may mean breeding pigs that have been given maternal antibodies against CSFV from sows vaccinated with CSFV vaccine. The definition of "CSFV vaccine" in this case will be described later. The giving of maternal antibodies is achieved by suckling colostrum. That is to say, "the breeding pigs of the second generation and later" may specifically mean breeding pigs that have been given maternal antibodies against CSFV by suckling colostrum from sows vaccinated with CSFV vaccine. The breeding pigs of the second generation and later may or may not be born from sows vaccinated with CSFV vaccine. Typically, the breeding pigs of the second generation and later may be born from sows vaccinated with CSFV vaccine. The sow that gives birth to the breeding pigs of the second generation and later and the sow that suckles colostrum to the breeding pigs of the second generation and later may or may not be the same individual. Typically, the sow that gives birth to the breeding pigs of the second generation and later and the sow that suckles colostrum to the breeding pigs of the second generation and later may be the same individual. That is to say, "the breeding pigs of the second generation and later" may typically mean breeding pigs that are born from sows vaccinated with CSFV vaccine and have been given maternal antibodies from the said sows. The breeding pigs of the second generation and later may be born more than 30 days after the vaccination of the CSFV vaccine by the sow vaccinated with CSFV vaccine. Also, the breeding pigs of the second generation and later may be suckled with colostrum more than 30 days after the vaccination of the CSFV vaccine by the sow vaccinated with CSFV vaccine. When the sow vaccinated with CSFV vaccine has been vaccinated with the CSFV vaccine two or more times, "after the vaccination of the CSFV vaccine" may mean after the first vaccination of the CSFV vaccine.

[0094] In the definition of breeding pigs from the second generation onwards, the "CSFV vaccine" refers to the CSFV vaccine inoculated to the sows of breeding pigs from the second generation onwards, and there is no particular limitation as long as it can induce immunity against CSFV. The CSFV vaccine inoculated to the sows of breeding pigs from the second generation onwards may be, for example, a live vaccine or an inactivated vaccine. Examples of the CSFV live vaccine include the CSFV live vaccines exemplified above. Examples of the CSFV inactivated vaccine include the CSFV E2 subunit vaccine. The inoculation of the CSFV vaccine to the sows of breeding pigs from the second generation onwards may be, for example, the first inoculation of the CSFV vaccine (also referred to as "primary mass vaccination") only when the breeding area of the sow is designated as an area recommended for CSFV vaccine inoculation. In addition, sows that have been inoculated with the CSFV vaccine may have enhanced immunity against CSFV by the method described in this specification (for example, the first method).

[0095] The inoculation mode of the subunit vaccine (for example, conditions related to inoculation such as the inoculation route, inoculation dose, number of inoculations, inoculation timing, etc. of the subunit vaccine) is not particularly limited as long as an immune induction effect can be obtained.

[0096] The subunit vaccine may be inoculated into pigs by injection. Examples of the inoculation route of the subunit vaccine include intramuscular inoculation and subcutaneous inoculation. The subunit vaccine may be inoculated into pigs as it is or after being appropriately prepared into a form suitable for inoculation into pigs. For example, when the subunit vaccine is provided in a form that is not a liquid such as a dried product, the subunit vaccine may be prepared into a liquid form with a solvent and then inoculated into pigs.

[0097] The number of doses of the subunit vaccine may be, for example, 1, 2 or more, or 3 or more. The number of doses of the subunit vaccine may be, for example, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. The number of doses of the subunit vaccine may be, for example, within a range defined by any combination of the ranges exemplified above. The number of doses of the subunit vaccine may particularly be 1, 2, or 3.

[0098] When the number of doses of the subunit vaccine is 2 or more, the implementation modes of each dose (for example, conditions related to vaccination such as the composition of the subunit vaccine, vaccination route, vaccination dose, etc.) may or may not be the same. When the number of doses of the subunit vaccine is 2 or more, the implementation modes of each dose may particularly be the same.

[0099] The vaccination dose of the subunit vaccine may be, for example, 10 μg / dose or more, 20 μg / dose or more, 50 μg / dose or more, 100 μg / dose or more, 200 μg / dose or more, or 500 μg / dose or more, when converted to the vaccination dose of the E2 subunit, and may be 1000 μg / dose or less, 500 μg / dose or less, 2 00 μg / dose or less, 100 μg / dose or less, 50 μg / dose or less, or 20 μg / dose or less, and may also be within a range defined by a non - conflicting combination thereof. Specifically, the vaccination dose of the subunit vaccine may be, for example, 10 - 20 μg / dose, 20 - 50 μg / dose, 50 - 100 μg / dose, 100 - 200 μg / dose, 200 - 500 μg / dose, or 500 - 1000 μg / dose when converted to the vaccination dose of the E2 subunit. Specifically, the vaccination dose of the subunit vaccine may be, for example, 10 - 1000 μg / dose, 20 - 500 μg / dose, or 50 - 200 μg / dose when converted to the vaccination dose of the E2 subunit.

[0100] The inoculation of the subunit vaccine may be carried out, for example, after 1 month or later, 2 months or later, 3 months or later, 4 months or later, 5 months or later, or 6 months or later after the first inoculation of the subunit vaccine, and may also be carried out until 12 months, 9 months, 6 months, 5 months, 4 months, or 3 months after the first inoculation of the subunit vaccine, and may be carried out within the period defined by their non - contradictory combinations. The inoculation of the subunit vaccine may be carried out, in particular, after 4 months or later after the first inoculation of the subunit vaccine. Specifically, the inoculation of the subunit vaccine may be carried out, for example, from 1 month to 2 months, 2 months to 3 months, 3 months to 4 months, 4 months to 5 months, 5 months to 6 months, 6 months to 9 months, or 9 months to 12 months after the first inoculation of the subunit vaccine.

[0101] The inoculation of the subunit vaccine may be carried out, for example, when the pig to be inoculated with the subunit vaccine is a sow, up to 3 months before, 2 months before, 1 month before, 2 weeks before, or 1 week before the sow gives birth to piglets. The inoculation of the subunit vaccine may be carried out, in particular, up to 1 week before the sow gives birth to piglets.

[0102] The inoculation of the subunit vaccine may be carried out, for example, when the pig to be inoculated with the subunit vaccine is a sow, up to 3 months before, 2 months before, 1 month before, 2 weeks before, or 1 week before the sow nurses the piglets with colostrum. The inoculation of the subunit vaccine may be carried out, in particular, up to 1 week before the sow nurses the piglets with colostrum.

[0103] In the first method, the immunity against CSFV in pigs may be induced to the desired degree. The induction of immunity against CSFV can be confirmed, for example, using the neutralizing antibody titer against CSFV as an index.

[0104] The neutralizing antibody titer against CSFV after subunit vaccine inoculation may be greater than the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation. The neutralizing antibody titer against CSFV after subunit vaccine inoculation may be, for example, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 7 times or more, 10 times or more, or 15 times or more the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation, and may be 100 times or less, 50 times or less, 20 times or less, or 10 times or less the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation, and may be within a range defined by any non - conflicting combination thereof. The neutralizing antibody titer against CSFV after subunit vaccine inoculation may particularly be 4 times or more the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation. Specifically, the neutralizing antibody titer against CSFV after subunit vaccine inoculation may be, for example, 1.5 to 100 times, 2 to 100 times, 3 to 100 times, 4 to 100 times, or 5 to 100 times the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation. The ratio of the neutralizing antibody titer against CSFV after subunit vaccine inoculation to the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation (= neutralizing antibody titer against CSFV after subunit vaccine inoculation / neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation) is also referred to as the "enhancement ratio of neutralizing antibody titer".

[0105] The neutralizing antibody titer against CSFV after subunit vaccine inoculation may be, for example, 0.8 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more as the S / P value of the ELISA method It may also be 2.2 or more, 2.4 or more, 2.6 or more, or 2.8 or more, and may also be 3 or less, 2.8 or less, 2.6 or less, 2.4 or less, 2.2 or less, 2 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, or 1 or less, and may be a range defined by any non - conflicting combination thereof. The neutralizing antibody titer against CSFV after subunit vaccine inoculation is specifically, for example, as the S / P value of the ELISA method, may be 0.8 - 1, 1 - 1.2, 1.2 - 1.4, 1.4 - 1.6, 1.6 - 1.8, 1.8 - 2, 2 - 2.2, 2.2 - 2.4, 2.4 - 2.6, 2.6 - 2.8, or 2.8 - 3. The neutralizing antibody titer against CSFV after subunit vaccine inoculation is specifically, for example, as the S / P value of the ELISA method, may be 0.8 - 3, 1 - 2.4, or 1 - 2.

[0106] The increase in neutralizing antibody titer by subunit vaccine inoculation (that is, the value obtained by subtracting the neutralizing antibody titer against CSFV at the time of subunit vaccine inoculation from the neutralizing antibody titer against CSFV after subunit vaccine inoculation) may be, for example, as the S / P value of the ELISA method, 0.4 or more, 0.6 or more, 0.8 or more, 1 or more, 1.2 or more, 1.4 or more, 1.6 or more, 1.8 or more, 2 or more, 2.2 or more, 2.4 or more, 2.6 or more, or 2.8 or more, and may also be 3 or less, 2.8 or less, 2.6 or less, 2.4 or less, 2.2 or less, 2 or less, 1.8 or less, 1.6 or less, 1.4 or less, 1.2 or less, 1 or less, 0.8 or less, or 0.6 or less, and may be a range defined by any non - conflicting combination thereof. The increase in neutralizing antibody titer by subunit vaccine inoculation is specifically, for example, as the S / P value of the ELISA method, may be 0.4 - 0.6, 0.6 - 0.8, 0.8 - 1, 1 - 1.2, 1.2 - 1.4, 1.4 - 1.6, 1.6 - 1.8, 1.8 - 2, 2 - 2.2, 2.2 - 2.4, 2.4 - 2.6, 2.6 - 2.8, or 2.8 - 3. The increase in neutralizing antibody titer by subunit vaccine inoculation is specifically, for example, as the S / P value of the ELISA method, may be 0.4 - 3, 0.6 - 2.4, or 0.8 - 2.

[0107] The neutralizing antibody titer against CSFV after subunit vaccine inoculation may be the value at any time point after subunit vaccine inoculation. The neutralizing antibody titer against CSFV after subunit vaccine inoculation may be, for example, the value at any time point after 7 days, 14 days, 21 days, 28 days, or 35 days after the inoculation of the subunit vaccine, and may also be the value at any time point up to 49 days, 42 days, 35 days, 28 days, or 21 days after the inoculation of the subunit vaccine, and may be the value at any time point within any non - conflicting combination of these periods. The neutralizing antibody titer against CSFV after subunit vaccine inoculation may particularly be the value at any time point between 14 days and 42 days after subunit vaccine inoculation. The neutralizing antibody titer against CSFV after subunit vaccine inoculation may more particularly be the value on the 28th day after subunit vaccine inoculation.

[0108] The method for measuring the neutralizing antibody titer against CSFV is not particularly limited as long as the neutralizing antibody titer against CSFV can be measured with the desired accuracy. The neutralizing antibody titer against CSFV can be measured, for example, by a conventional method. Examples of the method for measuring the neutralizing antibody titer include a neutralization test and an ELISA method. That is, the neutralizing antibody titer may be, for example, directly measured by a neutralization test or may be estimated by an ELISA method. For example, the neutralizing antibody titer can be estimated from the S / P value of the ELISA method (Kobayashi, Creation of a table for estimating neutralizing antibody titer based on the S / P value of the porcine fever ELISA, Porcine Disease Association Report No. 80, 2022, 12-16). The estimation of the neutralizing antibody titer from the S / P value of the ELISA method can be performed, for example, by ROC analysis. The measurement of the neutralizing antibody titer against CSFV by the ELISA method can be performed, for example, using a commercially available kit for measuring the neutralizing antibody titer against CSFV. Examples of such a kit include the Porcine Fever ELISA Kit II (Nippon Gene). The measurement of the neutralizing antibody titer against CSFV by the ELISA method can be performed, for example, as described in the examples. Note that "the enhancement ratio of the neutralizing antibody titer satisfies a certain condition" means that, unless otherwise specified, the enhancement ratio of the neutralizing antibody titer calculated by any arbitrary measurement method (which may be, for example, a neutralization test or an ELISA) satisfies the certain condition. For example, "the enhancement ratio of the neutralizing antibody titer is 4-fold or more" means that the enhancement ratio of the neutralizing antibody titer calculated by any arbitrary measurement method (which may be, for example, a neutralization test or an ELISA) is 4-fold or more.

[0109] In the first method, the variation in neutralizing antibody titers against CSFV may be suppressed by inoculation with a subunit vaccine. For example, the variation in neutralizing antibody titers against CSFV may be reduced after inoculation with a subunit vaccine as compared to that at the time of subunit vaccine inoculation. Also, for example, the variation in neutralizing antibody titers against CSFV may be reduced after inoculation with a subunit vaccine as compared to that after inoculation with a live CSFV vaccine when the live CSFV vaccine is inoculated instead of the subunit vaccine. In the first method, the variation in neutralizing antibody titers against CSFV may be suppressed to a desired degree. The suppression of the variation in neutralizing antibody titers against CSFV includes a decrease in the coefficient of variation (= standard deviation / mean value) of the neutralizing antibody titers against CSFV. The coefficient of variation of the neutralizing antibody titers against CSFV may be calculated, for example, when a plurality of individual pigs are inoculated with a subunit vaccine.

[0110] The coefficient of variation of the neutralizing antibody titers against CSFV after inoculation with a subunit vaccine may be, for example, 1 or less, 0.9 or less, 0.8 or less, 0.7 or less, 0.6 or less, 0.5 or less, 0.45 or less, 0.4 or less, 0.35 or less, 0.3 or less, 0.25 or less, or 0.2 or less. The coefficient of variation of the neutralizing antibody titers against CSFV after inoculation with a subunit vaccine may particularly be 0.3 or less.

[0111] The coefficient of variation of the neutralizing antibody titers against CSFV after inoculation with a subunit vaccine may be, for example, less than 1-fold, 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, or 0.2-fold or less of the coefficient of variation of the neutralizing antibody titers against CSFV at the time of subunit vaccine inoculation.

[0112] The coefficient of variation of the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine may be, for example, less than 1-fold, 0.9-fold or less, 0.8-fold or less, 0.7-fold or less, 0.6-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, or 0.2-fold or less of the coefficient of variation of the neutralizing antibody titer against CSFV after vaccination with the live CSFV vaccine when the live CSFV vaccine is inoculated instead of the subunit vaccine.

[0113] The coefficient of variation of the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine may be a value at any time point after vaccination with the subunit vaccine. The coefficient of variation of the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine may be, for example, a value at any time point after 7 days, 14 days, 21 days, 28 days, or 35 days after vaccination with the subunit vaccine, and may also be a value at any time point up to 49 days, 42 days, 35 days, 28 days, or 21 days after vaccination with the subunit vaccine, and may be a value at any time point within a period of any non-contradictory combination thereof. The coefficient of variation of the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine may particularly be a value at any time point between 14 days and 42 days after vaccination with the subunit vaccine. The coefficient of variation of the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine may more particularly be a value at 28 days after vaccination with the subunit vaccine.

[0114] Also, "the variation in the neutralizing antibody titer against CSFV is suppressed" may mean, for example, that the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine converges within the range of the neutralizing antibody titer against CSFV after vaccination with the subunit vaccine as exemplified above.

[0115] The second aspect of the method described in this specification is to prevent the infection of CSFV in pigs, in pigs A method for preventing the occurrence of CSF and / or alleviating the symptoms during the occurrence of CSF in pigs. Hereinafter, the same method (i.e., the second aspect of the method described in this specification) is also referred to as the "second method".

[0116] The second method includes inducing immunity against CSFV in pigs by the first method.

[0117] That is, by implementing the first method, specifically, by inducing immunity against CSFV by implementing the first method, the infection of CSFV in pigs may be prevented. Also, by implementing the first method, specifically, by inducing immunity against CSFV by implementing the first method, the occurrence of CSF in pigs may be prevented. Further, by implementing the first method, specifically, by inducing immunity against CSFV by implementing the first method, the symptoms during the occurrence of CSF in pigs may be alleviated.

[0118] The third aspect of the method described in this specification is a method for conferring immunity against CSFV to piglets. Hereinafter, the same method (i.e., the third aspect of the method described in this specification) is also referred to as the "third method".

[0119] The third method includes inducing immunity against CSFV in sows by the first method.

[0120] That is, by implementing the first method in sows, specifically, by inducing immunity against CSFV in sows by implementing the first method, immunity against CSFV may be conferred to piglets.

[0121] Immunity to CSFV in piglets may be conferred, for example, by the transfer of antibodies from sows to piglets. The transfer of antibodies to piglets is conferred by suckling from sows (especially suckling of colostrum). That is, the sow (i.e., the sow in which immunity to CSFV has been induced by the first method) may be the sow that suckles colostrum to the piglet (i.e., the piglet to which immunity is to be conferred in the third method). The sow that gives birth to the piglet and the sow that suckles colostrum to the piglet may or may not be the same individual. The sow that gives birth to the piglet and the sow that suckles colostrum to the piglet may typically be the same individual.

[0122] The third method may include suckling colostrum from the sow to the piglet.

[0123] In the third method, the variation in the transferred antibody titer against CSFV in piglets may be suppressed. That is, in one aspect, the "method of conferring immunity to CSFV on piglets" may be read as the "method of suppressing the variation in the transferred antibody titer against CSFV in piglets".

[0124] The "transferred antibody titer against CSFV in piglets" means the neutralizing antibody titer against CSFV transferred to piglets as transferred antibodies. The transferred antibody titer against CSFV in piglets can be measured, for example, by measuring the neutralizing antibody titer against CSFV in piglets. Also, the transferred antibody titer against CSFV in piglets can be estimated, for example, from the neutralizing antibody titer against CSFV in the sow that suckles colostrum to the piglets. The estimation of the transferred antibody titer against CSFV in piglets from the neutralizing antibody titer against CSFV in the sow can be carried out, for example, as described in the examples.

[0125] Specifically, the transferred antibody titer against CSFV in piglets may be estimated to be, for example, equivalent to the neutralizing antibody titer against CSFV in the sow that suckles colostrum to the piglets. Therefore, regarding the suppression of the variation in the transferred antibody titer against CSFV in piglets, for example, the description regarding the suppression of the variation in the neutralizing antibody titer against CSFV in the sow in the first method can be applied mutatis mutandis.

[0126] The period during which the multiplication factor of the maternally-derived antibody titer against CSFV in piglets is maintained at 32-fold or more may be, for example, 30 days or more, 40 days or more, 50 days or more, or 60 days or more, assuming that the half-life of the maternally-derived antibody titer is 10.1 days. The period during which the multiplication factor of the maternally-derived antibody titer against CSFV in piglets is maintained at 100-fold or more may be, for example, 20 days or more, 30 days or more, 40 days or more, or 50 days or more, assuming that the half-life of the maternally-derived antibody titer is 10.1 days. That is, the maternally-derived antibody titer against CSFV in piglets may be a value such that the period during which the multiplication factor of the maternally-derived antibody titer against CSFV in piglets is maintained at 32-fold or more or 100-fold or more falls within the range exemplified above.

[0127] Also, "the variation in the maternally-derived antibody titer against CSFV in piglets is suppressed" may mean, for example, that the maternally-derived antibody titer against CSFV in piglets converges within the range of the maternally-derived antibody titer against CSFV in piglets as exemplified above.

[0128] A fourth aspect of the method described in this specification is a method for preventing infection with CSFV in piglets, preventing the occurrence of CSF in piglets, and / or alleviating the symptoms at the time of occurrence of CSF in piglets. Hereinafter, the same method (that is, the fourth aspect of the method described in this specification) is also referred to as the "fourth method".

[0129] The fourth method includes conferring immunity against CSFV to piglets by the third method.

[0130] That is, by carrying out the third method, specifically, by conferring immunity against CSFV to piglets by carrying out the third method, infection with CSFV in piglets may be prevented. Also, by carrying out the third method, specifically, by conferring immunity against CSFV to piglets by carrying out the first method, the occurrence of CSF in piglets may be prevented. Further, by carrying out the first method, specifically, by conferring immunity against CSFV to piglets by carrying out the third method, the symptoms at the time of occurrence of CSF in piglets may be alleviated.

Example

[0131] The present invention will be further specifically described below with reference to non-limiting examples.

[0132] [Example 1] Based on the sequence information of the CSFV E2 subunit gene registered in GenBank (NIH gene sequence database), the region to be adopted as a vaccine antigen was determined, and the artificial synthesis of the gene was performed. Next, three constructs for expressing the E2 subunit were designed using the silkworm-baculovirus expression system.

[0133] The three constructs are as follows. The details are shown in Figure 1. (a) A construct in which a polynucleotide encoding the amino acid sequence of the E2 subunit found in the CSFV epidemic field strain (the amino acid sequence shown in SEQ ID NO: 2) is linked downstream of a polynucleotide encoding the secretion signal peptide of the silkworm 30K protein (b) In the above (a), a polynucleotide encoding a ZZ tag (a peptide linked with the IgG Fc binding region of Protein A) for promoting protein solubilization is linked downstream of a polynucleotide encoding the amino acid sequence of the E2 subunit found in the CSFV epidemic field strain (the amino acid sequence shown in SEQ ID NO: 2) (c) In the above (b), a construct in which a polynucleotide encoding the 30K protein secretion signal peptide is substituted with a polynucleotide encoding the secretion signal peptide of the E2 subunit found in the CSFV epidemic field strain

[0134] In addition, the abbreviations in Figure 1 are shown as follows. PH: Polyhedrin promoter SV40: SV40 polyadenylation signal B1: Recombination site for Gateway cloning B2: Recombination site for Gateway cloning 30K: Secretion signal peptide of Bombyx mori 30K protein CSF: Secretion signal peptide of E2 subunit found in CSFV epidemic field strains CSFV E2 1-331aa : E2 subunit found in CSFV epidemic field strains H8S: 8-Histidine (H8) tag ZZ: ZZ tag

[0135] First, three constructs were each constructed on plasmid pENTR11. These were named pENTR11L21-30K-CSFV-E2(A)-H8STREP, pENTR11L21-30K-CSFV-E2(A)-ZZH8STREP, and pENTR11IL21-NSP-CSFV-E2(A)-ZZ-H8STREP, respectively. These were each inserted onto the baculovirus transfer plasmid pFastBac by the Gateway reaction. After constructing pFastBacL21-30K-CSFV-E2(A)-H8STREP, pFastBacL21-30K-CSFV-E2(A)-ZZ-H8STREP, and pFastBacL21-NSP-CSFV-E2(A)-ZZH8STREP, they were each transformed into Escherichia coli strains carrying the gene-deficient Bacmid / BmNPV T3-Delta3P derived from the BmNPV T3 strain, and the Tn7 transfer reaction was induced in E. coli to construct the recombinant Bacmids, BacmidL21-30K-CSFV-E2(A)-H8STREP, BacmidL21-30K-CSFV-E2(A)-ZZ-H8STREP, and BacmidL21-NSP-CSFV-E2(A)-ZZ-H8STREP. After extracting these Bacmids, they were each introduced into the silkworm cultured cells BmN to obtain the recombinant baculoviruses, BmNPVL21-30K-CSFV-E2(A)-H8STREP, BmNPVL21-30K-CSFV-E2(A)-ZZH8STREP, and BmNPVL21-NSP-CSFV-E2(A)-ZZ-H8STREP. These recombinant baculoviruses were amplified three times using the silkworm cultured cells BmN to prepare high-titer stock solutions of each virus.

[0136] [Example 2] Each of the virus stock solutions was inoculated by injection of 10 μl into the 5th instar, 3-day-old silkworm larvae, and serum was collected 4 days later. When comparing the E2 subunit found in the CSFV epidemic field strain secreted in the serum, the expression of 30K-CSFV-E2(A)-ZZ-H8STREP and NSP-CSFV-E2(A)-ZZ-H8STREP, which are the products of constructs (b) and (c), was good.

[0137] [Example 3] Based on the results of the expression analysis, it was decided to purify 30K-CSFV-E2(A)-ZZ-H8STREP, which is the product of construct (b). BmNPVL21-30K-CSFV-E2(A)-ZZ-H8STREP was inoculated by injection of 10 μl into the 5th instar, 3-day-old larvae of the hybrid of Xa50 and n22, which is a protein highly expressing strain, and serum (10 ml × 2) was collected 4 days later. Blood cells and the like were separated from each 10 ml of the collected serum (about 25 portions) by centrifugation, and after collecting the supernatant, 40 ml of buffer A (20 mM Tris-HCl, 500 mM NaCl; pH 7.5, 1.0% NP-40) was added and ultrasonic treatment was performed. The baculovirus was inactivated using a surfactant (NP-40) for this solution, and subjected to affinity purification using two His TrapTM Excel columns. Next, the respective fractions were combined, concentrated using an Amicon (registered trademark) Ultra-15 mL centrifugal filter, and made up to 50 ml with buffer B (100 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA; pH 8.0). Thereafter, it was subjected to affinity purification using a Strep-Tactin Superflow column. The respective fractions were combined, concentrated, dialyzed, and then quantified. After the two-step affinity purification, concentration, dialysis, and quantification were performed. For dialysis, storage Buffer (10% trehalose, IxPBS(-), pH 7.4) was used. When quantification was performed using BSA as a standard, the concentration of the E2 subunit in the solution after dialysis was 0.41 mg / ml. Since a total of 17.6 ml of solution could be prepared, approximately 7.2 mg of the E2 subunit was recovered with high purity.

[0138] [Example 4] To confirm the removal and inactivation of recombinant baculovirus in the purified E2 subunit solution, 80 μl of the purified product was added to the culture medium of silkworm cultured cells BmN. Incidentally, the baculovirus is designed to also express EGFP, enabling highly sensitive detection of residual virus. Three days and four days after the addition of the purified product, when the fluorescence images were confirmed, compared with the Positive control, no fluorescence derived from EGFP was observed in the purified product addition group. Thus, it was confirmed that the recombinant baculovirus was removed and inactivated by ultrasonic treatment, surfactant treatment, and two-step affinity purification of the serum.

[0139] [Example 5] A vaccine was prepared using the purified E2 subunit prepared as described above as a vaccine antigen. Specifically, using PBS as a solvent, the purified E2 subunit (100 μg / dose), water-in-oil adjuvant (25 w / v%), and thimerosal (0.01 w / v% or less) were mixed to prepare a vaccine.

[0140] [Example 6] Ten fattening pigs vaccinated with live swine fever vaccine at about 3 weeks of age were introduced, and divided into two groups of 5 pigs each: a test group administered 2 mL per pig of the vaccine prepared above as a test substance, and a control group administered 2 mL per pig of physiological saline. Each test substance was administered at about 150 days of age. Using the day of administration of the test substance as day 0, blood was collected on days 0 and 14, and the antibody titer was measured using an ELISA kit (Swine Fever Eliza Kit II, Nippon Gene) with the serum as a specimen. The S / P values are shown in Table 1, and the graph thereof is shown in Figure 1. A significant difference was observed in the test group 14 days after immunization compared with the control group (p = 0.024).

[0141]

Table 1

[0142] <Description of the Sequence Listing> Sequence No.: 1: Nucleotide sequence encoding the E2 subunit found in the CSFV epidemic field strain 2: Amino acid sequence of the E2 subunit found in the CSFV epidemic field strain

Claims

1. A construct comprising a polynucleotide encoding an amino acid sequence for expressing a polypeptide consisting of the amino acid sequence of any one of the following (1), (2), or (3) using a baculovirus expression system: (1) The amino acid sequence of the E2 subunit found in a field strain of classical swine fever virus (CSFV) (SEQ ID NO: 2); (2) An amino acid sequence containing substitution, deletion, insertion, and / or addition of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and having a function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having a function of producing an antibody having neutralizing activity against CSFV.

2. The construct contains a polynucleotide encoding a secretion signal peptide, The polypeptide is one to which a secretion signal peptide is added, The construct according to claim 1.

3. The construct according to claim 2, wherein the secretion signal peptide is the secretion signal peptide of the E2 subunit found in a field strain of CSFV or the secretion signal peptide of the silkworm 30K protein.

4. The construct contains a polynucleotide encoding a soluble tag peptide, The polypeptide is one to which a soluble tag peptide is added, The construct according to claim 1.

5. The construct according to claim 4, wherein the soluble tag peptide is ZZ or SUMO.

6. The construct contains a polynucleotide encoding a tag peptide for protein purification, The polypeptide has a protein purification tag peptide added thereto. The construct according to claim 1.

7. The construct according to claim 6, wherein the protein purification tag peptide is a His tag or a Strep tag.

8. A recombinant baculovirus into which the construct according to claim 1 has been introduced.

9. A method for producing a polypeptide, comprising: The polypeptide is a polypeptide consisting of the amino acid sequence of any one of the following (1), (2), or (3), and a recovery step of recovering the polypeptide expressed in a baculovirus-susceptible insect infected with the recombinant baculovirus according to claim 8 from the insect. Production method: (1) The amino acid sequence of the E2 subunit found in the field strain of classical swine fever virus (CSFV) (SEQ ID NO: 2); (2) In the amino acid sequence shown in SEQ ID NO: 2, including substitution, deletion, insertion, and / or addition of 1 to 45 amino acid residues, and producing an antibody having neutralizing activity against CSFV An amino acid sequence having the function of; (3) An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having the function of producing an antibody having neutralizing activity against CSFV.

10. The production method according to claim 9, wherein the recovery step is a step of recovering the polypeptide from the serum of the insect.

11. The production method according to claim 9 or 10, wherein the insect is a silkworm.

12. A subunit vaccine against classical swine fever virus (CSFV), comprising: A subunit vaccine comprising a polypeptide consisting of the amino acid sequence of any one of the following (1), (2), or (3): (1) The amino acid sequence (SEQ ID NO: 2) of the E2 subunit found in a field strain of classical swine fever virus (CSFV); (2) An amino acid sequence that contains substitutions, deletions, insertions, and / or additions of 1 to 45 amino acid residues in the amino acid sequence shown in SEQ ID NO: 2 and has the function of producing an antibody having neutralizing activity against CSFV; (3) An amino acid sequence having 85% or more identity to the amino acid sequence shown in SEQ ID NO: 2 and having the function of producing an antibody having neutralizing activity against CSFV.

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