WO2022158453A1 - Ｗｓｓｖワクチン - Google Patents

Ｗｓｓｖワクチン Download PDF

Info

Publication number
WO2022158453A1
WO2022158453A1 PCT/JP2022/001638 JP2022001638W WO2022158453A1 WO 2022158453 A1 WO2022158453 A1 WO 2022158453A1 JP 2022001638 W JP2022001638 W JP 2022001638W WO 2022158453 A1 WO2022158453 A1 WO 2022158453A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino acid
acid sequence
seq
peptide
wssv
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/001638
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
龍洙 朴
ブーニャキダ ジラユ
シュ ジェン
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shizuoka University NUC
Original Assignee
Shizuoka University NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shizuoka University NUC filed Critical Shizuoka University NUC
Priority to JP2022576692A priority Critical patent/JPWO2022158453A1/ja
Publication of WO2022158453A1 publication Critical patent/WO2022158453A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a vaccine for preventing or treating infection with white spot syndrome virus (WSSV).
  • WSSV white spot syndrome virus
  • WSD White spot disease
  • WSSV White Spot Syndrome Virus
  • dsDNA supercoiled, circular double-stranded DNA
  • ORFs putative open reading frames
  • WSSV is also known to contain at least six virion proteins, namely VP19 and VP28 derived from envelope proteins, VP24 and VP26 derived from tegument proteins, and VP15 and VP664 derived from nucleocapsid proteins (Non-Patent Document 1, 2).
  • Shrimp infected with WSSV show symptoms such as lethargy, anorexia, reduced food intake, reduced repair activity, loose cuticles, reddish discoloration, and white calcified spots (0.5-3 mm in diameter) on the exoskeleton. is seen, resulting in a mortality rate of 90% within one week.
  • Non-Patent Documents 3, 4 viral protein subunits, attenuated WSSV and DNA/RNA-based immunological agents are known. Most of them are recombinant subunit vaccines such as VP19, VP24, VP26, VP28, VP292 and VP466, among which the use of VP28 has been reported (Non-Patent Documents 5-9). These WSSV subunit vaccines were expressed in E. coli.
  • the present inventor has so far investigated the anti-WSSV effect (WSSV infection-preventing effect) of five recombinant WSSV proteins VP15, VP19, VP24, VP26 and VP28. These proteins were expressed and purified using silkworms and Escherichia coli, and an in vivo antiviral assay using kuruma shrimp confirmed that the nucleocapsid protein VP15 exhibits high immunogenicity against WSSV infection (Non-Patent Document 10). .
  • the purpose of the present invention is to provide a vaccine that has a protective effect against white spot syndrome virus (WSSV) infection.
  • WSSV white spot syndrome virus
  • SR-11 SEQ ID NO: 7
  • a fragment peptide of WSSV protein VP15 has a high WSSV infection-preventing effect, and completed the present invention.
  • the present invention is as follows.
  • An antigenic peptide comprising the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having deletion, substitution or insertion of 1 to 3 amino acid residues in the amino acid sequence shown in SEQ ID NO: 7, wherein 8 to 30
  • WSSV white spot syndrome virus
  • the antigenic peptide consists of the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having deletion, substitution or insertion of 1 to 3 amino acid residues in the amino acid sequence shown in SEQ ID NO: 7 [1] Or the vaccine according to [2].
  • the antigenic peptide is a peptide containing 8 to 30 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, or 8 to 30 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1.
  • the vaccine according to [1] which is a peptide having 80% or more sequence identity with the peptide consisting of the base.
  • the diet according to [6] which is a health feed.
  • the vaccine of the present invention can prevent or treat white spot disease in crustaceans.
  • the antigenic peptide of the present invention is short (8 to 50 amino acid residues) and can be easily mass-produced in E. coli or silkworm, it is expected to be applied to cultured shrimp.
  • FIG. GST glutathione-S-transferase for affinity purification
  • FLAG tag FLAG tag for Western blot analysis (DYKDDDDK; SEQ ID NO: 10)
  • M molecular weight marker
  • S supernatant
  • P precipitate.
  • Example 1 the results of SDS-PAGE of fusion proteins of each VP-15 fragment peptide purified by GST affinity chromatography and GST are shown.
  • M molecular weight marker.
  • 2 is a graph showing the survival rate (%), which is an index of the protective effect against WSSV infection of each peptide fragment of WSSV-VP15 using kuruma shrimp in Example 1.
  • FIG. FIG. 2 is a schematic diagram showing the amino acid position and length of each fragment peptide (KR-11, SR-11, KK-13, SK-10) of VP15 (aa26-57) .
  • the survival rate (%) which is an index of the protective effect of each fragment peptide of VP15 (aa26-57) against WSSV infection, is shown using kuruma shrimp.
  • (a) is a graph showing protective effect by KR-11, SR-11, SK-10 and KK-13
  • (b) is a graph showing protective effect by SR-11.
  • 3 shows the results of confirming the expression of GST-VP15 or GST-VP15 (aa25-36) in silkworm larvae and pupae by Western blotting in Example 3.
  • FIG. 4 shows the result of confirming the interaction between VP15, VP15 (aa26-57) and SR-11 and the receptor in Example 4 by Western blotting.
  • Example 5 the relative survival rate (%), which is an index of the protective effect against WSSV infection after administration of feed containing various vaccines using kuruma shrimp, is shown.
  • FIG. 6 shows the results of examining changes in mRNA expression levels of Dorsal gene (a) and Relish gene (b) after administration of various vaccine-containing feeds using kuruma shrimp in Example 6.
  • FIG. 10 shows the results of examining changes in mRNA expression levels of STAT gene (a) and ProPO gene (b) after administration of vaccine-containing feed using kuruma shrimp in Example 6.
  • the vaccine of this embodiment is a vaccine for preventing or treating infection with white spot syndrome virus (WSSV), and has 1 to 3 antigenic peptides comprising amino acid sequences having deletions, substitutions or insertions of amino acid residues of 8 to 30 amino acid residues.
  • the antigenic peptide means a peptide having antigenicity (immunogenicity), particularly a peptide having antigenicity, particularly WSSV antigenicity, in aquatic crustaceans such as shrimp.
  • VP15 (SEQ ID NO: 1) is an 80 amino acid protein with a very high pI value, showing homology to DNA binding proteins of eukaryotic origin and baculovirus p6.9. VP15 contains 20-21% each of serine, arginine and lysine. Although VP15 is the major nucleocapsid protein involved in packaging the viral genome into capsids, its crystal structure is still unknown.
  • a peptide (full length: 11 amino acid residues) consisting of the amino acid sequence shown in SEQ ID NO: 7 is a VP15 fragment peptide consisting of the 37th to 47th amino acid residues of the VP15 protein, and is herein referred to as "SR-11". is sometimes called.
  • SR-11 When SR-11 is administered to shrimp, it induces immunity and has a preventive effect against WSSV infection.
  • SR-11 has WSSV antigenicity (WSSV immunogenicity) and functions as a WSSV vaccine, allowing protection against WSSV infection.
  • the antigenic peptide in this embodiment is the amino acid sequence shown in SEQ ID NO: 7, or an amino acid having deletion, substitution or insertion of 1 to 3 amino acid residues in the amino acid sequence shown in SEQ ID NO: 7. including sequences (sometimes referred to herein as "SR-11 variants"). That is, the antigenic peptide in this embodiment includes, at least in part, SR-11 or an SR-11 variant.
  • SR-11 mutants are not particularly limited as long as they have WSSV antigenicity, but deletion or substitution of 1, 2 or 3 amino acid residues at any position in the amino acid sequence of SR-11 or may have an insert. In the case of 2 or 3 amino acid residues, they may be consecutive amino acid residues or discontinuous amino acid residues.
  • Amino acids to be substituted or inserted may be natural or non-natural.
  • Natural amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L -methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
  • Group 1 leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
  • Group 2 aspartic acid, glutamic acid, isoaspartic acid, Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid group 3: asparagine, glutamine group 4: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid group 5: proline, 3 - Hydroxyproline, 4-Hydroxyproline Group 6: Serine, Threonine, Homoserine Group 7: Phenylalanine
  • the antigenic peptide in this embodiment consists of the above-described SR-11 or SR-11 variant, or contains the above-described SR-11 or SR-11 variant as a part, and has WSSV antigenicity.
  • the length is not particularly limited, but preferably consists of 8 to 30 amino acid residues, more preferably 8 to 25, 8 to 20, 9 to 20, 10 to 20, 9 to 15 , 10-15, 9-13, or 10-12 amino acid residues.
  • the amino acid residues in the portion other than SR-11 or SR-11 mutant in the antigenic peptide are not particularly limited, and the WSSV antigenicity of SR-11 or SR-11 mutant Preferably, it is an amino acid residue that does not affect WSSV antigenicity or improves WSSV antigenicity.
  • the antigenic peptide in this embodiment may also be in the form of a fusion protein formed with a protein tag used for purification of the antigenic peptide.
  • tags include His-tag, Flag-tag, myc-tag, glutathione-S-transferase (GST), maltose-binding protein (MBP), Strep-tag, etc. These tags have short amino acid sequences.
  • the fusion protein with the antigenic peptide may be used as a vaccine as it is, since it does not affect the WSSV antigenicity.
  • the antigenic peptide and protein tag may be separated after purification.
  • the antigenic peptide in this embodiment preferably consists of SR-11 or SR-11 mutants. That is, the antigenic peptide has the amino acid sequence shown in SEQ ID NO: 7, or deletion or substitution of 1 to 3, preferably 2, more preferably 1 amino acid residue in the amino acid sequence shown in SEQ ID NO: 7. Alternatively, it preferably consists of an amino acid sequence having an insertion, and particularly preferably consists of the amino acid sequence shown in SEQ ID NO:7.
  • the antigenic peptide containing SR-11 or SR-11 mutant is a peptide consisting of 8 to 30 consecutive amino acid residues of the amino acid sequence shown in SEQ ID NO: 1, or SEQ ID NO: 8 to 30 amino acid residues having a sequence identity of 80% or more, preferably 85% or more, 90% or more, or 95% or more with a peptide consisting of consecutive amino acid residues of the amino acid sequence shown in 1. It may be a peptide consisting of The protein consisting of the amino acid sequence shown in SEQ ID NO: 1 is the VP15 protein, and the peptide consisting of the 37th to 47th amino acid residues of the VP15 protein is SR-11.
  • the antigenic peptide in this embodiment comprises SR-11 or SR-11 variants as all or part of the sequence. That is, the antigenic peptide in this embodiment is a fragment peptide or fragment peptide variant of the VP15 protein, which contains SR-11 or SR-11 variant as a whole or part of the sequence.
  • the antigenic peptides in this embodiment may be artificially synthesized, they can be mass-produced by expressing them in transformants or recombinant silkworms, which will be described later.
  • the antigenic peptide of interest may or may not be purified.
  • the recombinant silkworm may be dried as it is and mixed with feed for use.
  • the vaccine of this embodiment may consist of only an effective amount of the antigenic peptide, or may contain other components than the effective amount of the antigenic peptide.
  • the dosage form of the vaccine of this embodiment may be, for example, liquid, powder (freeze-dried powder, dry powder), granules, and the like.
  • Components other than antigenic peptides may be carriers commonly used in vaccine manufacture, including, for example, saline, buffered saline, dextrose, water, glycerol, isotonic aqueous buffers, and combinations thereof.
  • emulsifiers, preservatives (eg, thimerosal), tonicity agents, pH adjusters, inactivators (eg, formalin), etc. may be added as appropriate.
  • the vaccine of this embodiment can be obtained by mixing the antigenic peptide with other components such as a carrier and a component that enhances antigenicity, if necessary.
  • the vaccine of the present embodiment is intended to be administered to crustaceans, for example, farmed crustaceans, especially crustaceans suffering from white spot disease, preferably aquatic crustaceans such as shrimp and lobster, Vietnamese crustaceans such as shrimp and lobster, Vietnamesemp include, among others, Marsupenaeus japonicus, Litopenaeus vannamei, Penaeus monodon, and Fenneropenaeus indicus.
  • White spot disease can be effectively prevented or treated by administering the vaccine of this embodiment to a crustacean.
  • the administration route of the vaccine of this embodiment is not particularly limited, and may be intramuscular injection or oral administration, for example.
  • a single administration is effective, but multiple administrations, such as 2, 3, 4, or 5 administrations, provide a high infection-preventing effect.
  • It is also preferred to administer regularly for example 1 to 25 times a year, especially 3 times a year, 4 times a year, 6 times a year, 12 times a year or 1 to 4 times a month.
  • the administration interval is not particularly limited, and may be administered, for example, at intervals of 1 day to 3 months, 1 week to 2 months, 10 to 30 days, or 15 to 25 days.
  • oral administration it is mixed with food and given at regular intervals (e.g., once every 3 months, once every 2 months, once every month, once every 10 days, once every 5 days). or daily).
  • the dosage of the vaccine of this embodiment may vary depending on the administration frequency and administration route. preferably 10-20 ⁇ g antigenic peptide/g body weight or 10-20 ⁇ g antigenic peptide/g body weight.
  • the peptide of this embodiment comprises the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence having 1 to 3 amino acid residue deletions, substitutions, or insertions in the amino acid sequence shown in SEQ ID NO: 7, and , is an isolated peptide having 8-30 amino acid residues.
  • the peptide of this embodiment may be an antigenic peptide as described above.
  • the diet of this embodiment comprises an effective amount of the vaccine or peptide of this embodiment.
  • the diet is preferably a diet for crustaceans, and the effective amount of the vaccine or peptide may be 1-100 ⁇ g antigenic peptide/day/g body weight, and 5-50 ⁇ g antigenic peptide/day/day.
  • g body weight preferably 10-45 ⁇ g antigenic peptide/g body weight, or 10-20 ⁇ g antigenic peptide/day/g body weight.
  • Effective amounts may vary with dosing intervals and may be adjusted accordingly. For example, an effective amount for administration once every five days may be 1.5 to 3 times the effective amount for daily administration, preferably twice.
  • the diet of this embodiment may contain, in addition to the vaccine or peptide of this embodiment, dietary components suitable for crustaceans.
  • the diet of the present embodiment may contain, for example, proteins, lipids, carbohydrates, vitamins and minerals, and more specifically, fish meal, krill meal, wheat flour, corn, alfalfa meal, brewer's yeast.
  • vitamins (choline chloride, vitamin E, vitamin C , inositol, vitamin B5 , vitamin B2, Vitamin A , vitamin B1 , vitamin B6, vitamin B3, folic acid, vitamin D3 , biotin), minerals (Ca, Fe, Mg, Zn, Mn, Cu, I) may be included.
  • the diet may be a commercially available feed to which the vaccine or peptide of the present embodiment is added.
  • the vaccine or peptide of the present embodiment is expressed in transformants or recombinant silkworms described later.
  • the dry powder of the transformant or the recombinant silkworm may be used as it is without purifying the target antigenic peptide.
  • a diet of one embodiment may be a health feed containing the vaccine or peptide of this embodiment.
  • the diet of this embodiment is given to crustaceans at regular intervals (e.g., once every 3 months, once every 2 months, once every month, once every 10 days, once every 5 days). , or daily) can effectively prevent or treat white spot disease.
  • the nucleic acid molecule of this embodiment is a peptide consisting of the amino acid sequence shown in SEQ ID NO: 7 or an amino acid sequence having deletion, substitution or insertion of 1 to 3 amino acid residues in the amino acid sequence shown in SEQ ID NO: 7. and encodes an antigenic peptide having 8-30 amino acid residues.
  • the nucleic acid molecule of this embodiment can be obtained, for example, by subjecting the wild-type VP15 gene encoding the amino acid sequence shown in SEQ ID NO: 1 to error-prone PCR or the like as a template. Alternatively, it can be obtained by introducing site-directed mutation.
  • the nucleic acid molecule of this embodiment can also be obtained by artificial synthesis of a nucleic acid molecule encoding an amino acid sequence designed based on the amino acid sequence of SEQ ID NO: 1 or 7.
  • the nucleic acid molecule obtained as described above is used as it is or is cleaved with an appropriate restriction enzyme or the like, incorporated into a vector by a conventional method, and the obtained recombinant expression vector is introduced into a host cell.
  • the base sequence of the nucleic acid molecule can be determined by analysis using a base sequence analyzer such as the method.
  • Vectors that can be used for determining the nucleotide sequence of nucleic acid molecules include pFastBac, pGEX-6P-1, and the like.
  • the nucleic acid molecule of this embodiment may contain a nucleic acid that encodes the aforementioned protein tag in addition to the nucleic acid that encodes the antigenic peptide of this embodiment. In this case, it is preferable to allow the antigenic peptide of this embodiment and the protein tag to be expressed as a fusion protein.
  • the transformant of this embodiment can be obtained by transforming a host cell with the nucleic acid molecule of this embodiment.
  • the host cell is not particularly limited as long as it can express the antigenic peptide of the present embodiment, but examples include prokaryotes such as E. coli, yeast, insect cells, plant cells, and animal cells. Escherichia coli, yeast, insect cells or mammalian cultured cells are preferred.
  • the nucleic acid molecule of the present embodiment as it is or a recombinant expression vector obtained by incorporating the nucleic acid molecule of the present embodiment into a vector is used to transform host cells. Conversion is preferred.
  • the vector that can be used for the recombinant expression vector is not particularly limited, and can be appropriately selected depending on the host cell.
  • vectors include pGEX-6P-1 and the like.
  • vectors include pYES2 and the like.
  • vectors include pcDNA3.1(+) and the like.
  • a transformant can be obtained by introducing the above nucleic acid molecule or recombinant expression vector into a host cell. Introduction may be performed by a conventional method.
  • the antigenic peptide of the present embodiment is expressed, collected, and optionally purified to obtain the antigenic peptide of the present embodiment. of antigenic peptides can be obtained.
  • the recombinant silkworm of this embodiment can be obtained by incorporating the nucleic acid molecule of this embodiment.
  • the nucleic acid molecule of the present embodiment can be incorporated into silkworms as it is or by using a recombinant expression vector obtained by incorporating the nucleic acid molecule of the present embodiment into a vector.
  • Vectors for integration into silkworms include, for example, pFastBac.
  • a recombinant silkworm is preferably used for the production of the antigenic peptide of this embodiment.
  • Silkworms can be bred if there is a space where the temperature can be controlled to some extent, and since they feed on mulberry leaves or artificial feed, they do not require control devices such as bioreactors, culture media, and fermenters, and production costs are low.
  • the protein expression level per silkworm is said to be comparable to the expression level of 30 ml of E. coli culture solution, and it is possible to obtain proteins with high quality such as three-dimensional structure and modification.
  • pupae As silkworms, larvae, preferably 5th instar larvae may be used, and pupae may be used. In the case of silkworms, purification costs are high, but in the case of oral administration, pupae may be used from the viewpoint that recombinant silkworms can be directly administered as feed without purifying antigenic peptides. In addition, it may be a pupa from the viewpoint of being rich in protein and lipid and excellent in nutrition.
  • Methods for incorporating the nucleic acid molecule of this embodiment into silkworms include the mNPV Bacmid expression method, which can express an antigenic peptide. Also, after expression, antigenic peptides can be affinity recovered by GST tag or FLAG tag. It can then be purified if desired.
  • Example 1 Screening of WSSV Antigens Showing Anti-WSSV Effect (1) Expression of Fragment Peptide of WSSV-VP15 As shown in FIG. Peptide (VP15 (aa1-25) ; SEQ ID NO: 2), peptide consisting of 26th to 57th amino acid residues (VP15 (aa26-57) ; SEQ ID NO: 3), peptide consisting of 58th to 80th amino acid residues (VP15 ( aa58-80) ; SEQ ID NO: 4), and a peptide (VP15 (aa1-25, 58-80) ; SEQ ID NO: 5) in which the 1st to 25th amino acid residues and the 58th to 80th amino acid residues are directly linked Fragmented into 4 pieces.
  • a DNA encoding a fusion protein was prepared by fusing a FLAG tag (DYKDDDDK; SEQ ID NO: 10) to the C-terminus of WSSV-VP15 (wsv214), and the expression vector pGEX-6P-1 ( GE Healthcare, USA) to form a fusion protein with GST at the C-terminus of GST (glutathione-S-transferase, 26 kDa) (Non-Patent Document 10).
  • This constructed plasmid was named "pGEX-VP15".
  • plasmids expressing VP-15 and each of the above four fragment peptides were constructed. Specifically, inverse PCR was performed using KOD-PLUS-NEO (Toyobo, Japan) and the primer set shown in Table 1. In Table 1, each primer set is a primer set for amplifying the plasmid fragment expressing the fragment peptide described on the left side, FW is the forward primer, and RV is the reverse primer. Inverse PCR was performed after denaturation at 94°C for 2 minutes, followed by 30 cycles of 98°C for 10 seconds, 57°C for 30 seconds, 68°C for 2 minutes and 30 seconds, and finally extension at 68°C for 7 minutes.
  • the amplified product was treated with T4 DNA polymerase (NEB, Japan) and then self-ligated.
  • T4 DNA polymerase NEB, Japan
  • the resulting plasmid containing each peptide fragment was transformed into E. coli DH5 ⁇ by the heat shock method.
  • the transformed E. coli was inoculated onto an agar medium containing 50 ⁇ g/ml ampicillin, plasmids were extracted from ampicillin-positive colonies, and the sequences of each fragment were confirmed by Sanger DNA sequencing.
  • the culture was chilled on ice for 30 minutes and protein expression was determined by adding isopropyl- ⁇ -thiogalactopyranoside (IPTG) to the culture to a final concentration of 0.5 mM. was induced and incubation continued for 18 hours at 16°C. After 18 hours, E. coli were harvested by centrifugation (6,000 ⁇ g, 4° C., 15 minutes), washed twice with phosphate-buffered saline (PBS, pH 7.3), and kept at ⁇ 80° C. until use. saved with
  • IPTG isopropyl- ⁇ -thiogalactopyranoside
  • E. coli was resuspended in PBS containing 1 ⁇ proteinase inhibitor (cOmpleteTM, Mini, EDTA-free Protease Inhibitor Cocktail, Sigma-Aldrich, Japan) and 10 ⁇ g/ml lysozyme.
  • the suspension was sonicated on ice (70% amplitude, 30 sec on/off, 15 cycles), centrifuged (10,000 ⁇ g, 4° C., 10 min) and passed through a 0.2 ⁇ m cellulose acetate membrane (Minisart (registered trademark) NML, Sartorius, Japan).
  • FIG. 2(a) shows the results of SDS-PAGE
  • FIG. 2(b) shows the results of Western blotting.
  • the molecular weight of the fusion protein of VP-15 and GST is 37.0 kDa, and the molecular weight of each VP-15 fragment peptide and each fusion protein of GST is 30.8 kDa (VP15 (aa1-25) ), 31 6 kDa (VP15 (aa26-57) ), 30.5 kDa (VP15 (aa58-80) ) and 33.5 kDa (VP15 (aa1-25, 58-80) ).
  • the expression of each fusion protein was confirmed because the molecular weight of each fusion protein shown in FIG. 2 agreed with the estimated molecular weight.
  • FIG. 3 shows the results of SDS-PAGE (12% SDS gel) of the purified fusion protein.
  • control shrimp were also inoculated with 10 ⁇ L/tail of PBS in the PBS group and 10 ⁇ g/g shrimp body weight of GST-VP15 (aa26-57) in the control GST group in the same manner as the experimental group.
  • a control group of shrimp was also injected intramuscularly with PBS or GST in the same manner as the experimental group.
  • WSSV was obtained as follows. A 10 ⁇ 3 dilution of virus prepared from juvenile shrimp infected with natural WSSV was injected intramuscularly into adult tiger shrimps (average body weight 78 g), hemolymph was collected 3 days after infection and stored at -80°C. Prior to each experiment, stored virus was thawed, centrifuged at 1500 xg for 10 minutes at 4°C, and the resulting supernatant diluted in PBS for challenge.
  • Kuruma prawns were reared in dechlorinated electrolyzed seawater (33.05 ⁇ 0.13 ppt) at 24 ⁇ 1.8°C using double-bottom tanks on a sand bed. They were fed commercial shrimp diet (Juveniles P-2, Maruha, Japan) at 3% of body weight per day.
  • the survival rate (%) of each group was obtained by the formula below.
  • the 20-day survival rate of each group is shown in FIG.
  • the PBS control group and the GST control group had a dramatic decrease in survival from day 4 to day 7, reaching 20% and 33.3% survival respectively at 20 days post-challenge.
  • the VP15 (aa1-25) administration group showed the same tendency as the GST group.
  • VP15, VP15 (aa58-80) , or VP15 (aa1-25, 58-80) administration groups showed survival rates of 42.1%, 47.6%, and 38.9% at day 20, respectively. .
  • VP15 (aa26-57) showed a survival rate of 57.9% at 20 days and was the only experimental group showing a significant difference ( ⁇ 2 test, p ⁇ 0:05) ( Fig . 4). .
  • Example 2 Identification of Antigenic Domain in WSSV-VP15 (1) Peptide Synthesis Example 1 revealed that VP15 (aa26-57) has higher antigenicity than other peptide fragments. Therefore, VP15 (aa26-57) was further fragmented to identify the antigenic domain.
  • VP15 (aa26-57) was further subdivided into four peptide fragments: KR-11 (KTKSRRGSKKR, SEQ ID NO:6), SR-11 (STTAGRISKRR, SEQ ID NO:7), KK-13 (KRRSPSMKKRAGK, SEQ ID NO:7). 8), and SK-10 (SPSMKKRAGK, SEQ ID NO: 9), respectively, which were commercially synthesized (GL Biochem Ltd., China).
  • HPLC high performance liquid chromatography
  • ESI-MS electrospray ionization mass spectrometry
  • HPLC was used for purification of each peptide using an Inertsil ODS-SP column (purity >95%).
  • the purity and molecular weight of the purified synthetic peptides were analyzed using liquid chromatography-electrospray ionization coupled with mass spectrometry (LC-MS/ESI, Agilent-6125B).
  • Example 3 Expression of VP15 and SR-11 in Silkworm Using pGEX-VP15 as a template and using the primer set described in Table 1, VP15 (SEQ ID NO: 1) or SR-11 fragment peptide of VP15 (SEQ ID NO: 1) or SR- A pFastBac vector incorporating a nucleic acid molecule encoding GST-VP15 or SR-11 fused with GST and Flag tag such that 11 (SEQ ID NO: 7) is downstream of GST and upstream of Flag tag. made. Furthermore, a recombinant BmNPV bacmid for silkworm expression was prepared by transforming the pFastBac vector into E.
  • BmNPV bacmid DNA was mixed with DMRIE-C (Thermo Fisher Scientific, Japan) at a concentration of 0.2 ⁇ g BmNPV bacmid/1 ⁇ L, and the resulting solution was injected into 5-instar silkworm larvae or larvae at 20 ⁇ L/mouse with a needle 26-gauge syringe. injected into the pupae. )
  • Proteins were detected using a mouse anti-Flag tag antibody (1:10000, MBL, Japan) as the primary antibody for Western blotting and an anti-mouse antibody (1:10000) as the secondary antibody (MBL, Japan).
  • a mouse anti-Flag tag antibody (1:10000, MBL, Japan)
  • an anti-mouse antibody (1:10000)
  • MBL mouse anti-Flag tag antibody
  • VP15, VP15 (aa26-57) , and SR-11 are nucleic acid molecules encoding fusion proteins fused to GST and Flag tags such that they are downstream of GST and upstream of Flag tag, respectively. It was constructed and expressed in E. coli. As a control, GST alone was used. On the other hand, the receptor MjgCqR-His was expressed in silkworms in the same manner as described above.
  • Example 5 Evaluation of Infection-Protecting Effect by Oral Administration of Various Vaccines
  • purified liquid 1 and powder containing various proteins/peptides shown in Table 2 were prepared. 2 to 6 were used.
  • the GST protein was expressed in E. coli in the same manner as in Example 1 and purified with a GST affinity column to obtain purified solution 1 (negative control, 2 mg protein/mL).
  • GST protein was expressed in silkworm pupae in the same manner as in Example 3, was not purified after expression, was lyophilized and ground in a mortar to obtain powder 2 (negative control).
  • Fusion proteins of antigenic peptides of various purposes (VP15, VP15 (aa26-57) or SR-11) and GST were similarly expressed in silkworm pupae, not purified after expression, freeze-dried, and ground in a mortar. Powders 3-5 were obtained. SR-11 was artificially synthesized in the same manner as in Example 2 to obtain powder 6. The content of the target antigenic peptide (VP15, VP15 (aa26-57) , or SR-11) in the obtained powders 3 to 5 was quantified and shown in Table 2.
  • Purified liquid 1 and powders 2 to 6 were mixed with a commercially available feed (Juveniles P-2, Maruha, Japan) so as to have the composition shown in Table 3 to prepare a total of 29.8 g of mixed feed. 0.5% of spreading agent (Shering-Plough Animal Health, USA) was added to this mixed feed. After that, 1788 ⁇ L of PBS buffer corresponding to 6% of the mixed feed was mixed to obtain vaccine-containing feeds 1 to 6. Vaccine-containing diets 1 and 2 served as negative controls. Feed (PBS feed) prepared in the same manner without the addition of various proteins/peptides was also used as a negative control.
  • the GST-VP15 group, the GST-VP15 (aa26-57) group, the GST-SR-11 group, and the SR-11 group, the prawns were fed vaccine-containing feed 3 to 6 in an amount of 1.3 g / day. and fed for 23 days. After that, the animals were returned to the normal feed, continued to be bred for 6 days, and tested for WSSV infection on the 30th day.
  • GST (derived from E. coli) group, GST (derived from silkworm pupae) group and PBS group were administered vaccine-containing feeds 1 to 2 and PBS feed, respectively, in the same manner as in the experimental group.
  • the WSSV infection test adopted the immersion attack test.
  • Shrimp killed by WSSV infection were added with 9 times the body weight of PBS, ground, centrifuged at 1500 xg for 15 minutes at 4°C, and the resulting supernatant was diluted to a predetermined DNA copy number. was diluted with PBS to obtain a WSSV homogenate.
  • the WSSV homogenate was diluted 10-2.8 with water so as to give 1.27 ⁇ 10 6 DNA copies/g shrimp when the total weight of the challenged shrimp was 30 g/water (L). added and soaked for 2 hours.
  • the shrimps subjected to the test were taken out into a net, washed with clean seawater under running water for 3 minutes, and housed in a breeding tank.
  • FIG. 9 shows the relative survival rate of each group 20 days after the WSSV infection test.
  • the relative survival rates 20 days after the WSSV infection test were 60.3% and 68.8%, respectively.
  • the GST-VP15 group had a relative survival rate of 81.1%, while the GST-VP15 (aa26-57) group had a relative survival rate of 100%. This demonstrates that VP15 (aa26-57) has a high protective effect against WSSV infection.
  • the relative survival rate was 72.2%, which was higher than the relative survival rate of the GST group derived from E. coli and silkworm pupae, but lower than the relative survival rate of the GST-VP15 group. .
  • the SR-11 group also showed a high relative survival rate of 90.5%. Although the relative survival rate of the GST-SR-11 group was lower than that of the GST-VP15 group, the relative survival rate of the SR-11 group was higher than that of the GST-VP15 group. SR-11 alone was shown to be more effective in protecting against WSSV infection than the form containing SR-11. Since both the GST-VP15 group and the GST-VP15 (aa26-57) group had a relative survival rate of 80% or more, various proteins (vaccine) in the experimental group were also effective in protecting shrimp from WSSV infection by oral administration. It is considered to be a target.
  • Example 6 Changes in Gene Expression Levels Due to Administration of Vaccine-Containing Feed
  • various genes that is, transcription factors that play a central role in NF- ⁇ B immune responses. Changes in the expression levels of the Dorsal gene and Relish gene, the STAT gene that induces a signal in the antiviral response and improves the expression of AMP, and the ProPO gene that induces the expression of a protein that blocks the invasion of pathogens before and after administration. evaluated.
  • RNA was extracted from the gills of prawns using NucleoSpin RNA (Macherey-Nagel, Germany). .
  • the extracted total RNA was reverse transcribed using ReverTra Ace qPCR RT Master Mix (Toyobo, Japan) to synthesize first-strand cDNA.
  • ReverTra Ace qPCR RT Master Mix Toyobo, Japan
  • quantitative real-time PCR qPCR
  • FIGS. 10 and 11 The relative expression level of mRNA of each gene is a value relative to the expression level of the ⁇ -actin gene. Also, the expression level of the ⁇ -actin gene was defined as "1".

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Biomedical Technology (AREA)
  • Epidemiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Communicable Diseases (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
PCT/JP2022/001638 2021-01-20 2022-01-18 Ｗｓｓｖワクチン Ceased WO2022158453A1 (ja)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2022576692A JPWO2022158453A1 (https=) 2021-01-20 2022-01-18

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021006994 2021-01-20
JP2021-006994 2021-01-20

Publications (1)

Publication Number Publication Date
WO2022158453A1 true WO2022158453A1 (ja) 2022-07-28

Family

ID=82549419

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/001638 Ceased WO2022158453A1 (ja) 2021-01-20 2022-01-18 Ｗｓｓｖワクチン

Country Status (2)

Country Link
JP (1) JPWO2022158453A1 (https=)
WO (1) WO2022158453A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024147326A1 (ja) * 2023-01-06 2024-07-11 住友化学株式会社 エビ目の生物におけるウイルス感染症に対する予防または治療用経口投与組成物

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003506338A (ja) * 1999-08-03 2003-02-18 アクゾ・ノベル・エヌ・ベー 白点症候群ウイルス由来のタンパク質及びその使用
JP2008063302A (ja) * 2006-09-11 2008-03-21 Fisheries Research Agency クルマエビ属ホワイトスポット病用経口ワクチン
WO2011004697A1 (ja) * 2009-07-09 2011-01-13 国立大学法人宮崎大学 甲殻類急性ウイルス血症に対するワクチン

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003506338A (ja) * 1999-08-03 2003-02-18 アクゾ・ノベル・エヌ・ベー 白点症候群ウイルス由来のタンパク質及びその使用
JP2008063302A (ja) * 2006-09-11 2008-03-21 Fisheries Research Agency クルマエビ属ホワイトスポット病用経口ワクチン
WO2011004697A1 (ja) * 2009-07-09 2011-01-13 国立大学法人宮崎大学 甲殻類急性ウイルス血症に対するワクチン

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BOONYAKIDA JIRAYU, XU JIAN, SATOH JUN, NAKANISHI TAKAFUMI, MEKATA TORU, KATO TATSUYA, PARK ENOCH Y.: "Antigenic properties of VP15 from white spot syndrome virus in kuruma shrimp Marsupenaeus japonicus", FISH & SHELLFISH IMMUNOLOGY, ACADEMIC PRESS, LONDON,, GB, vol. 101, 1 June 2020 (2020-06-01), GB , pages 152 - 158, XP055952901, ISSN: 1050-4648, DOI: 10.1016/j.fsi.2020.03.061 *
SATOH J, NISHIZAWA T, YOSHIMIZU M: "Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28", DISEASES OF AQUATIC ORGANISMS, INTER-RESEARCH, DE, vol. 82, 20 November 2008 (2008-11-20), DE , pages 89 - 96, XP055952903, ISSN: 0177-5103, DOI: 10.3354/dao01978 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024147326A1 (ja) * 2023-01-06 2024-07-11 住友化学株式会社 エビ目の生物におけるウイルス感染症に対する予防または治療用経口投与組成物

Also Published As

Publication number Publication date
JPWO2022158453A1 (https=) 2022-07-28

Similar Documents

Publication Publication Date Title
Yi et al. Construction of a DNA vaccine and its protective effect on largemouth bass (Micropterus salmoides) challenged with largemouth bass virus (LMBV)
Xue et al. Oral vaccination of BacFish-vp6 against grass carp reovirus evoking antibody response in grass carp
JP4731907B2 (ja) 受動免疫のための蛋白質およびペプチド発現
Cui et al. Identification and functional characterization of an interferon regulatory factor 7-like (IRF7-like) gene from orange-spotted grouper, Epinephelus coioides
US20040047881A1 (en) Delivery of disease control in aquaculture and agriculture using microbes containing bioactive proteins
Wei et al. Molecular cloning and characterization of a new G-type lysozyme gene (Ec-lysG) in orange-spotted grouper, Epinephelus coioides
Gonzalez-Silvera et al. Recombinant nodavirus vaccine produced in bacteria and administered without purification elicits humoral immunity and protects European sea bass against infection
Jha et al. Protection of Procambarus clarkii against white spot syndrome virus using recombinant oral vaccine expressed in Pichia pastoris
Gao et al. Plasmid pcDNA3. 1-s11 constructed based on the S11 segment of grass carp reovirus as DNA vaccine provides immune protection
Citarasu et al. Oral vaccination of Macrobrachium rosenbergii with baculovirus-expressed M. rosenbergii nodavirus (MrNV) capsid protein induces protective immunity against MrNV challenge
KR101782692B1 (ko) 능성어의 신경괴사증 바이러스(nnv) 백신 제조방법
Li et al. Recombinant baculovirus BacCarassius-D4ORFs has potential as a live vector vaccine against CyHV-2
KR20230164694A (ko) 경구 백신 조성물
JP2025503468A (ja) 魚類心筋炎を予防するための組成物及び方法
Li et al. CsTNF1, a teleost tumor necrosis factor that promotes antibacterial and antiviral immune defense in a manner that depends on the conserved receptor binding site
Jia et al. Probiotics surface-delivering Fiber2 protein of fowl adenovirus 4 stimulate protective immunity against hepatitis-hydropericardium syndrome in chickens
Boonyakida et al. Identification of antigenic domains and peptides from VP15 of white spot syndrome virus and their antiviral effects in Marsupenaeus japonicus
WO2022158453A1 (ja) Ｗｓｓｖワクチン
Zhang et al. TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish
Boonyakida et al. Immunostimulation of shrimp through oral administration of silkworm pupae expressing VP15 against WSSV
Zhao et al. iTRAQ proteome analysis of insight into TLR20. 2 functions through IFN1 signaling exerts regulatory effects on GCRV replication
EP3003346B1 (en) Fish vaccine
EP2906704B1 (fr) Novirhabdovirus recombinant utilisable comme vecteur d'antigènes
ES2788923T3 (es) Nuevo bacteriófago y composición que comprende al mismo
Yang et al. CgATP synthase β subunit involved in the regulation of haemocytes proliferation as a CgAstakine receptor in Crassostrea gigas

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22742575

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022576692

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22742575

Country of ref document: EP

Kind code of ref document: A1