WO2011004697A1 - Vaccin contre une virémie aiguë des crustacés - Google Patents

Vaccin contre une virémie aiguë des crustacés Download PDF

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Publication number
WO2011004697A1
WO2011004697A1 PCT/JP2010/060503 JP2010060503W WO2011004697A1 WO 2011004697 A1 WO2011004697 A1 WO 2011004697A1 JP 2010060503 W JP2010060503 W JP 2010060503W WO 2011004697 A1 WO2011004697 A1 WO 2011004697A1
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vaccine
crustacean
protein
wssv
gene
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PCT/JP2010/060503
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English (en)
Japanese (ja)
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正博 酒井
利明 伊丹
智哉 河野
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国立大学法人宮崎大学
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Priority to JP2011521875A priority Critical patent/JP5649188B2/ja
Publication of WO2011004697A1 publication Critical patent/WO2011004697A1/fr

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    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/195—Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/18011—Nimaviridae
    • C12N2710/18022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/18011—Nimaviridae
    • C12N2710/18034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish
    • Y02A40/818—Alternative feeds for fish, e.g. in aquacultures

Definitions

  • the present invention relates to a vaccine effective for preventing viral infection in crustaceans.
  • Crustacean acute viremia also called white spot syndrome, vitiligo disease, etc.
  • white spot syndrome also called white spot syndrome, vitiligo disease, etc.
  • This disease is highly lethal within a few days after infection, and no effective prevention / treatment method has been established. Therefore, it is currently the only effective measure against this disease that the pathogenic virus, White Spot Syndrome Virus (WSSV), is not brought into the farm.
  • WSSV White Spot Syndrome Virus
  • this pathogenic virus WSSV has a wide host range in crustaceans (Non-patent Document 1), and almost no genetic variation is observed between isolated strains (Non-patent Document 2). There is also a high risk that WSSV infection in crustaceans will spread to other species of crustaceans farmed nearby. Therefore, the development of a simple and more effective prevention method for this disease is eagerly desired.
  • Patent Documents 1 and 2 include WSSV structural proteins as vaccines, or live attenuated bacteria or virus vectors into which genes encoding such structural proteins have been introduced as vaccines. A method of administration to crustaceans is disclosed.
  • DNA vaccines are not infectious and can be maintained in vivo for long periods of time to elicit both humoral and cellular immune responses, and are easy to manufacture and store.
  • DNA vaccines there is often a problem that an immune response cannot be elicited effectively depending on the administration method.
  • the present inventors administered a DNA vaccine prepared by incorporating a gene encoding a structural protein of WSSV virus into a pTargeTTM vector, which is a DNA vector for mammalian expression, to prawns.
  • a DNA vaccine prepared by incorporating a gene encoding a structural protein of WSSV virus into a pTargeTTM vector, which is a DNA vector for mammalian expression, to prawns.
  • An object of the present invention is to provide a vaccine that is effective in preventing crustacean acute viremia and exhibits an excellent preventive effect.
  • the present inventors administered a structural protein encoded by a specific gene among the structural protein genes of WSSV virus as a vaccine to a crustacean.
  • the inventors have found that blood can be prevented very effectively, and have completed the present invention.
  • the present invention includes the following.
  • WSSV white spot syndrome virus
  • a crustacean acute viremia preventive comprising the vaccine according to any one of (1) to (3) as an active ingredient.
  • a method for preventing crustacean acute viremia comprising administering the vaccine according to any one of (1) to (3) above to a crustacean.
  • the vaccine according to the present invention can provide crustaceans with high protection against white spot syndrome virus and can prevent crustacean acute viremia.
  • FIG. 6 is a characteristic diagram showing a photograph showing the result of SDS-PAGE of synthesized rVP28.
  • FIG. 5 is a characteristic diagram showing a calibration curve created using a quantitative value of ⁇ -lactalbumin. It is a characteristic figure which shows the survival rate of the prawn which was infected with WSSV 7 days after inoculation of the injection vaccine which concerns on this invention. It is a characteristic figure which shows the survival rate of the prawn which was infected with WSSV 7 days after the vaccination produced by the E. coli expression system. It is a characteristic figure which shows the survival rate of the prawn which was infected with WSSV 7 days after administration of the oral vaccine which concerns on this invention. It is a characteristic view which shows the design position of the primer and probe which were used for real-time PCR.
  • the vaccine according to the present invention is prepared by using the VP28 protein encoded by the VP28 gene among the genes encoding the structural proteins of white spot syndrome virus (WSSV) as an antigen, and the crustacean acute viral blood according to the present invention.
  • the prevention method of the disease is a method using the vaccine.
  • the vaccine of the present invention contains VP28 protein encoded by VP28 gene among them as an antigen.
  • the open reading frame sequence contained in the VP28 gene is shown in SEQ ID NO: 1, and the amino acid sequence encoded by the sequence is shown in SEQ ID NO: 2.
  • the full-length sequence of the VP28 protein serving as an antigen protein may be a partial sequence of a structural protein that is cleaved at the end but retains antigenicity.
  • the VP28 protein serving as the antigen protein consists of an amino acid sequence in which, for example, 1 to 100 (preferably 1 to 10) amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2, and It may be a protein or peptide that retains antigenicity.
  • WSSV structural protein genes many base sequences are registered in well-known base sequence databases such as international base sequence databases and GenBank.
  • base sequence databases such as international base sequence databases and GenBank.
  • VP15 gene AY374120, AY249451; VP28 gene: AY324881, AY249443; VP26 gene: AY249438, AY249439; VP19 gene: AY316119, AY249444; VP24 gene: AY249457, AY249458; VP35 gene: AY325896, VP14 Gene: AY422226 etc.
  • DNA amplification including VP28 gene is performed by PCR using a specific primer pair designed to sandwich the gene region from the information of the base sequence of VP28 gene using genomic DNA extracted from WSSV by a conventional method as a template
  • the product can be obtained.
  • mutation in a base sequence by well-known site-directed mutagenesis methods including Kunkel method and Gapped-duplex method.
  • Site-directed mutagenesis can also be performed using commercially available products such as Mutan-K C, Mutan -Super Express Km (Takara Bio), TAKARA LA PCRTM in vitro Mutagenesis kit (Takara Bio). .
  • the obtained DNA amplification product may be purified by any purification method known to those skilled in the art, such as an anion exchange chromatography method.
  • VP28 protein can be obtained according to a conventional method using the obtained VP28 gene.
  • the obtained VP28 gene is incorporated into a protein expression vector, this vector is introduced into a host cell, and the VP28 protein expressed in the obtained host cell is isolated and purified. Can be obtained.
  • a protein synthesis fraction derived from wheat germ a protein synthesis fraction derived from E. coli, a protein synthesis fraction derived from rabbit reticulocytes, and a protein synthesis fraction derived from insect cells were used.
  • Systems are known and can be used as appropriate.
  • the VP28 protein can be produced using a cell-free protein synthesis system using a protein synthesis fraction derived from wheat germ.
  • the vaccine according to the present invention is preferably synthesized using a cell-free protein synthesis system using a protein synthesis fraction derived from wheat germ, and an unpurified VP28 protein is used as an active ingredient.
  • the VP28 protein obtained as described above can be used as an active ingredient of a vaccine for preventing crustacean acute viremia caused by WSSV infection.
  • the VP28 protein as an active ingredient may be isolated and purified by any purification method known to those skilled in the art, such as an anion exchange chromatography method.
  • a cell-free protein synthesis system using a protein synthesis fraction derived from wheat germ it is preferably used as a vaccine without purification.
  • “vaccine” means a subunit vaccine preparation containing an antigen protein as an active ingredient.
  • the vaccine in the present invention may be a vaccine composition containing a pharmaceutically acceptable carrier (for example, which can be added to a pharmaceutical product or a marine product formulation) or an additive.
  • Carriers and additives include water, isotonic solutions, aqueous buffers, acceptable organic solvents and surfactants, auxiliaries, stabilizers, antioxidants, preservatives, and the like.
  • carriers and additives include, but are not limited to, sterile water, saline, Ringer's solution, PBS, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, Sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and other liposomes Artificial cell structures such as These carriers and additives are appropriately selected according to the administration route, dosage form, storage form, and the like of the vaccine.
  • the vaccine of the present invention may further contain an adjuvant, but since it originally has a high immunostimulatory function, it is not always necessary to add an additional adjuvant.
  • the vaccine of the present invention may further contain other
  • the vaccine according to the present invention exhibits a high effect on the prevention of crustacean acute viremia when administered to crustaceans.
  • crustacean acute viremia means a crustacean infection caused by white spot syndrome virus (WSSV) (also called white spot syndrome, leukosis etc.). This disease is typically characterized by white spots appearing on the body surface and death at an extremely high rate.
  • prevention of crustacean acute viremia means that WSSV that has entered the living body is excluded and / or its growth is suppressed, and as a result, the development of crustacean acute viremia is prevented or It means to reduce the incidence significantly.
  • the preventive effect of crustacean acute viremia obtained by administration of the vaccine of the present invention is typically indicated by an increase in survival rate (decrease in mortality rate) in the presence of WSSV.
  • the crustacea to be administered with the vaccine may be any crustacea that can be infected with WSSV, such as shrimp, crab, clam, crayfish, krill, daphnia, etc. Can be mentioned.
  • Particularly suitable examples of these crustaceans include, but are not limited to, decapod crustaceans, such as the shrimps, shrimps, lobsters, kingfishes, red lobsters, lobsters, lobsters, crayfishes And other organisms belonging to the genus, such as the Decapoda crabs, spider crabs, crabs, crabs, crabs, and crabs.
  • crustaceans suitable for administration include, but are not limited to, organisms in the family Penaeidae, such as Farfantepenaeus, Fenneropenaeus, Litopenaeus, Marsupenaeus, Melicertus, Metapenaeopsis, Metapenaeus, Penacheus, Examples include shrimp belonging to the genus Xiphopenaeus.
  • edible shrimp include shrimp (Marsupenaeus japonicus), southern tiger shrimp (Melicertus canaliculatus), tiger shrimp (Black tiger) (Penaeus monodon), tiger shrimp (Penaeus chinensis), ), Yellow shrimp (Penaeus latisulcatus), Indian shrimp (Fenneropenaeus indicus), reed shrimp (Metapenaeus ensis), horseshoe shrimp (Metapenaeus intermedius), and the like, but are not limited thereto.
  • the vaccine according to the present invention can be administered to crustaceans by any appropriate administration method.
  • the vaccine of the present invention can be administered to crustaceans, for example, but not limited to, by injection, immersion, spraying, or orally. More preferably, the vaccine of the present invention can be administered to the crustacean by intramuscular, intraperitoneal injection or the like, or orally administered to the crustacean in a form such as being included in feed.
  • the dose of the vaccine is preferably 0.1 to 50 ⁇ g, more preferably 1 to 10 ⁇ g of protein per animal to be administered.
  • the vaccine may be administered only once, but may be repeated two or more times at intervals.
  • the vaccine according to the present invention when the vaccine according to the present invention is orally administered, it is preferable to prepare a preparation suitable for oral administration.
  • the vaccine according to the present invention is preferably mixed with a feed ingredient to form a solid preparation.
  • the vaccine according to the present invention can be administered to the crustacea while giving a normal feed.
  • feed when feed is supplied to crustaceans, since feed is usually put into seawater, it is preferable to prevent VP28 protein, which is an active ingredient, from leaking into seawater before crustaceans feed.
  • a feed containing VP28 protein as an active ingredient preferably contains a spreading agent in addition to the feed ingredient.
  • the spreading agent is a substance that maintains a state in which the VP28 protein is attached to other feed ingredients, and is sometimes called a thickener.
  • the spreading agent conventionally known substances can be used without any limitation.
  • casein, sodium alginate, chitosan, cyclodextrin and the like can be used as a spreading agent.
  • the spreading agent in the feed is preferably 3% by weight or more. If the spreader is contained in an amount of 3% by weight or more, the VP28 protein must be administered to crustaceans because the original shape can be maintained for several hours after feeding the feed into seawater. Can do.
  • heat treatment is performed to apply high temperature to the feed so that the feed can maintain its shape in seawater.
  • a spreading agent is included in the feed containing the vaccine according to the present invention, the shape can be maintained in seawater without performing a heat treatment at a high temperature. That is, by using a spreading agent in the feed containing the vaccine according to the present invention, problems such as denaturation of the vaccine due to heat treatment can be avoided. In addition, the cost required for the heat treatment can be reduced by not performing the heat treatment.
  • the administration of the vaccine according to the present invention elicits both a humoral immune response and a cellular immune response against the VP28 protein in the crustacean organism.
  • the use of VP28 protein as an antigen protein has succeeded in obtaining a particularly excellent immunostimulatory effect.
  • crustacean acute viremia can be very effectively prevented by the induction of such an immune response and the remarkable immune activation effect.
  • the vaccine of the present invention has a remarkably high immunity enhancing effect as compared with a conventional vaccine using a conventional viral protein, attenuated virus, modified virus or the like as a vaccine antigen. Therefore, the method for preventing crustacean acute viremia by administering the vaccine of the present invention can be used very advantageously in, for example, the crustacean aquaculture industry.
  • PCR PCR was performed using the extracted DNA as a template and primers VP28-Fw (GGATCCATGGATCTTTCTTTCAC (SEQ ID NO: 3)) and VP28-Rv (ACTAGTTTACTCGGTCTCAGTGC (SEQ ID NO: 4)) specific to the VP28 gene.
  • primers VP28-Fw GGATCCATGGATCTTTCTTTCAC (SEQ ID NO: 3)
  • VP28-Rv ACTAGTTTACTCGGTCTCAGTGC (SEQ ID NO: 4)
  • PCR was performed using Gene Taq (NIPPON GENE).
  • the reaction solution was DW (28.5 ⁇ l), 10 ⁇ Gene Taq Universal Buffer (15 mmol / l Mg 2+ ) (5 ⁇ l), dNTP Mixture (2.5 mmol / each) (5 ⁇ l), Fw Primer (5 ⁇ M) (5 ⁇ l), Rv Primer (5 ⁇ M) (5 ⁇ l), Gene Taq (5 units / ⁇ l) (0.5 ⁇ l), and Template (1 ⁇ l) were mixed, and the reaction was performed in a volume of 50 ⁇ l.
  • the reaction conditions were: denaturation reaction at 94 ° C for 3 minutes, denaturation reaction at 94 ° C for 30 seconds, annealing reaction at 60 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute for 35 cycles, and finally 72 ° C The extension reaction was performed for 5 minutes. PCR reaction was performed using C1000 Thermal Cycler (BIORAD).
  • PCR product was ligated using pGEM T Easy Vector Systems (PROMEGA). Mix 5 ⁇ T4 DNA Ligase Buffer (5 ⁇ l), T Easy vector (50 ng / ⁇ l) (1 ⁇ l), T4 DNA Ligase (3 Weiss units / ⁇ l) (1 ⁇ l), and PCR product (3 ⁇ l) for 16 hours at 4 ° C. Reacted.
  • T4 DNA Ligase Buffer 5 ⁇ l
  • T Easy vector 50 ng / ⁇ l
  • T4 DNA Ligase 3 Weiss units / ⁇ l) (1 ⁇ l
  • PCR product 3 ⁇ l
  • the ligation product was transformed into TAM competent cells (ACTIFMOTIF) by the heat shock method.
  • Ligation product (3 ⁇ l) was added to TAM competent cell (50 ⁇ l) and allowed to stand on ice for 30 minutes. After reacting at 42 ° C. for 45 seconds, the mixture was allowed to stand on ice for 2 minutes.
  • the total amount of the bacterial solution was added to SOC medium (INVITROGEN) (450 ⁇ l), and cultured with shaking at 37 ° C. for 90 minutes.
  • the culture solution was inoculated into MacConkey agar medium (manufactured by SIGMA) containing 50 ⁇ g / ml ampicillin and cultured at 37 ° C. for 16 hours. After culture, positive clones were screened by the red-white collection.
  • Colony PCR The insert was confirmed by colony PCR. Colony PCR was performed using Gene Taq. DW (14.6 ⁇ l), 10 ⁇ Gene Taq Universal Buffer (15 mmol / l Mg 2+ ) (2 ⁇ l), dNTP Mixture (2.5 mmol / each) (1.6 ⁇ l), Fw-primer (5 ⁇ M) (0.8 ⁇ l), Rv- Primer (5 ⁇ M) (0.8 ⁇ l) and Gene Taq (0.2 ⁇ l) were mixed. Colonies were scraped and suspended in the reaction solution with a sterilized toothpick.
  • reaction conditions were 94 ° C for 3 minutes, denaturation at 94 ° C for 30 seconds, annealing at 60 ° C for 30 seconds, extension reaction at 72 ° C for 1 minute for 30 cycles, and finally 5 cycles at 72 ° C.
  • a minute extension reaction was performed.
  • PCR reaction was performed using C1000 Thermal Cycler (BIORAD). After completion of the reaction, 1.5% agarose gel electrophoresis was performed to confirm the presence or absence of a band.
  • Sequencing 1-7-1 Sequence Reaction A sequence reaction was performed using SP6 (ATTTAGGTGACACTATAGAA (SEQ ID NO: 5)) or T7 (TAATACGACTCACTATAGGG (SEQ ID NO: 6)) primer using the plasmid DNA obtained in 1-6 as a template. First, plasmid DNA (4 ⁇ l) was preheated for 1 minute at 95 minutes. Next, a primer (1.6 pmol / ⁇ l) (2 ⁇ l), DTCS Quick Start Master Mix (manufactured by Beckman coulter) (2 ⁇ l) and DW (2 ⁇ l) were added and reacted. The reaction conditions were 96 ° C for 20 seconds, 50 ° C for 20 seconds, and 60 ° C for 4 minutes for 30 cycles. The sequence reaction was performed using C1000 Thermal Cycler (manufactured by BIORAD).
  • Electrophoresis The whole amount of the sample purified in 1-7-2 was transferred to a CEQ sample plate (manufactured by Beckman coulter), and 1 drop of mineral oil (manufactured by Beckman coulter) was added.
  • a CEQ sample plate was set on a CEQ8000 Automated Sequencer (Beckman coulter) and electrophoresed.
  • 1% agarose L gel [Agarose-L Gel (1.5 g), 0.5 ⁇ TBE buffer (100 ml), ethidium bromide (10 ng / ⁇ l) (10 ⁇ l)] was electrophoresed. A band of the desired size was cut out from the gel, and DNA was extracted using a QIAquick Gel Extraction Kit (QIAGEN) according to the protocol. Next, it was ligated to the multiple cloning site of pEU-E01-MCS vector.
  • T4 DNA Ligase Buffer 5 ⁇ T4 DNA Ligase Buffer (5 ⁇ l), restriction enzyme-treated pEU-E01-MCS vector (1 ⁇ l), T4 DNA Ligase (3 Weiss units / ⁇ l) (1 ⁇ l), DNA excised from restriction enzyme-treated plasmid DNA (1 ⁇ l) ) And DW (2 ⁇ l) were mixed and reacted at 4 ° C. for 16 hours.
  • the sequence of this plasmid DNA was confirmed by the methods 1-4 and 7, and it was confirmed that the VP28 gene was incorporated into the protein expression vector.
  • VP28 recombinant protein A VP28 recombinant protein (rVP28) was prepared using a wheat germ cell-free protein synthesis system (CELLFREE SCIENCE). At the same time, control (Wheat) was prepared by adding DW instead of plasmid DNA. First, pre MIX (225 ⁇ l) was added to plasmid DNA (1 ⁇ g / ⁇ l) (25 ⁇ l), and a transcription reaction was performed at 37 ° C. for 6 hours. The transcription reaction was performed using C1000 Thermal Cycler (manufactured by BIORAD). After the reaction, agarose gel electrophoresis was performed to confirm the amount of mRNA synthesis.
  • CELLFREE SCIENCE wheat germ cell-free protein synthesis system
  • the protocol specifies that the concentration is 1 mg / ml and the purity is OD260nm / 280nm ratio of 1.70 to 1.85, but in this experiment the concentration is 1 mg / ml and the purity is OD260nm / 280nm.
  • the ratio was defined as 1.75 to 1.80. In particular, when the OD ratio was around 1.85, the transcription efficiency of mRNA was sometimes lowered. Therefore, in this experiment, 1.75 to 1.80 was determined as the optimal purity of plasmid DNA.
  • a translation reaction was performed by the multi-layer method.
  • MRNA (250 ⁇ l) synthesized by transcription reaction, WEPRO1240 (250 ⁇ l) and Creatine Kinase (20 mg / ml) (1 ⁇ l) were mixed.
  • 1 ⁇ SUB-AMIX (5.5 ml) was added to 6 WelllatePlate (manufactured by IWAKI), and the mixed solution was layered on the lower layer of 1 ⁇ SUB-AMIX.
  • RVP28 was synthesized by capping with parafilm and carrying out a translation reaction at 22 ° C. for 24 hours.
  • Results 3-1 Injection vaccine Fig. 3 shows the results of an experiment in which an injection vaccine was administered. From the results shown in FIG. 3, the survival rate of each group was 91.3% in the vaccine group (rVP28), 47.8% in the control 1 group (PBS), and 34.8% in the control 2 group (Wheat). The survival rate of rVP28 was significantly higher than that of control, and the RPS (vaccine efficacy rate) was 91.7% for PBS and 93.3% for Wheat. Therefore, the effectiveness of the vaccine against WSSV was confirmed 7 days after vaccination. The RPS (%) was calculated as 1 ⁇ (mortality in the vaccine administration group / mortality in the control group) ⁇ 100.
  • rVP28 was prepared using E. coli as a host cell, and the results of inoculating the subunit vaccine produced in the same manner are shown in FIG. From the results shown in FIG. 4, the mortality rate of rVP28 produced by the E. coli expression system was very high compared to the group inoculated with PBS alone. This is thought to be a result of E. coli endotoxin being toxic to shrimp, and that the post-vaccination endotoxin could not withstand the attack by WSDV. For this reason, when producing a vaccine using an E. coli expression system, it has been found that a purification treatment is necessary to remove components that are toxic to shrimp.
  • TaqMan Universal PCR Master Mix (12.5 ⁇ l), TaqMan MGB Probe (2.5 ⁇ l), Fw-Primer (10 pmol/ ⁇ l) (2.25 ⁇ l), Rv-Primer (10 pmol/ ⁇ l) (2.25 ⁇ l), DW (4.5 ⁇ l) was mixed, and 25 ⁇ l was dispensed into 96 well plates (Applied Biosystems).
  • the lid was covered with ABI PRISM Optical Adhesive Cover (Applied Biosystems) and set in 7300 Real-time PCR System (Applied Biosystems) for reaction.
  • the reaction conditions were maintained at 50 ° C. for 2 minutes, then heated at 95 ° C. for 10 minutes, and the reaction was started by a hot start method. Then, 40 cycles of annealing at 95 ° C. for 15 seconds and extension reaction at 60 ° C. for 1 minute were performed.
  • the vaccine and the method for preventing crustacean acute viremia according to the present invention can be used as an effective preventive measure against crustacean viremia that causes enormous damage in the aquaculture industry.

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  • Epidemiology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Feed For Specific Animals (AREA)
  • Farming Of Fish And Shellfish (AREA)
  • Fodder In General (AREA)

Abstract

La présente invention concerne un vaccin efficace pour la prévention d'une virémie aiguë des crustacés. L'invention concerne spécifiquement un vaccin destiné à la prévention d'une virémie aiguë des crustacés, qui utilise la protéine VP28 codée par le gène VP28 d'un virus de la maladie des points blancs (WSSV) en tant qu'antigène.
PCT/JP2010/060503 2009-07-09 2010-06-22 Vaccin contre une virémie aiguë des crustacés WO2011004697A1 (fr)

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JP2009-162802 2009-07-09
JP2009162802 2009-07-09

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WO2011004697A1 true WO2011004697A1 (fr) 2011-01-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022158453A1 (fr) * 2021-01-20 2022-07-28 国立大学法人静岡大学 Vaccin contre le wssv

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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
JP2005538721A (ja) * 2002-09-16 2005-12-22 アドバンスド バイオニュートリション コーポレーション 受動免疫のための蛋白質およびペプチド発現
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

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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
JP2005538721A (ja) * 2002-09-16 2005-12-22 アドバンスド バイオニュートリション コーポレーション 受動免疫のための蛋白質およびペプチド発現
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

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Title
SATOH, J. ET AL.: "Protection against white spot syndrome virus (WSSV) infection in kuruma shrimp orally vaccinated with WSSV rVP26 and rVP28", DIS AQUAT ORGAN, vol. 82, no. 2, 2008, pages 89 - 96 *
WITTEVELDT, J. ET AL.: "Protection of Penaeus monodon against white spot syndrome virus by oral vaccination", J VIROL, vol. 78, no. 4, 2004, pages 2057 - 61, XP003015737, DOI: doi:10.1128/JVI.78.4.2057-2061.2004 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022158453A1 (fr) * 2021-01-20 2022-07-28 国立大学法人静岡大学 Vaccin contre le wssv

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JPWO2011004697A1 (ja) 2012-12-20

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