JP2005112726A - Dna vaccine against viral hemorrhagic sepsticemia of flatfish - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA vaccine for fishes in order to induce protective immunity for viral hemorrhagic septicemia of a flatfish. <P>SOLUTION: This DNA vaccine comprises a nucleotide sequence encoding an immunogenic polypeptide against viral hemorrhagic sepsticemia virus of the flatfish or an expression vector containing the DNA as an active ingredient. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention relates to a DNA vaccine for inducing protective immunity against infectious diseases of fish of the Japanese flounder viral hemorrhagic septicemia virus (Viral Hemorrhagic Septicemia Virus; VHSV).
As used herein, “flounder viral hemorrhagic septicemia” means virulent hemorrhagic sepsis caused by infection with flounder VHSV, and thus not only includes viral hemorrhagic sepsis that developed in flounder, Viral hemorrhagic sepsis developed in fish other than flounder [for example, fish species belonging to the flounder family or the flounder family (for example, flounder or flounder) or sweetfish or black sea bream].

In the aquaculture industry of many aquatic organisms such as seafood, viral and bacterial diseases in aquaculture areas that are closed systems exist in high density of individuals. It is a big problem in the aquaculture industry.
Viral hemorrhagic sepsis has long been known as a viral disease that causes industrial damage to cultured rainbow trout in European countries. The causative virus has been found in many salmonid fishes and wild marine fishes, and has become a problem that threatens marine aquaculture (Non-Patent Document 1).
Viral hemorrhagic sepsis virus belongs to the Rhabdoviridae family and is an enveloped single-stranded RNA virus. Symptoms of viral hemorrhagic sepsis include body color blackening, eyeball protrusion, abdominal distension, anemia, and hemorrhage of the body side / base. In addition, hepatic and renal fullness / swelling / scarring and skeletal muscle punctate bleeding are observed (Non-patent Document 2). Further, histopathologically, renal necrosis of the urinary system and hematopoietic tissue, partial necrosis of the liver and spleen, and skeletal muscle bleeding are prominent.

From March to May 1996, it became clear that the mass death of cultured flounder that occurred in the sea surface split ginger under Kagawa Prefecture was due to viral hemorrhagic sepsis. Until now, viral hemorrhagic sepsis has been known in salmonids, but not in marine fish, and it has been clarified that this disease occurs only in marine fish. Since 1998, flounder viral hemorrhagic septicemia has gradually expanded, and in 2000, it also occurred in flounder farms in several prefectures along the Seto Inland Sea coast and the Bungo Channel coast. It has become. The disease develops at a water temperature of 8-15 ° C from December to May, and the cumulative mortality rate may exceed 70% in breeding fish, and it may be annihilated in small fish. Therefore, Japanese flounder viral hemorrhagic sepsis has been positioned as the first new disease since the enactment of the method for ensuring continuous aquaculture production (Non-patent Document 3). Further, the flounder virus hemorrhagic septic virus strain described in the present specification is considered to have a different group from the salmonid virus strains known in taxonomy (Non-Patent Document 4). ). As described above, flounder viral hemorrhagic sepsis is an important disease which is an industrial problem.
Most seafood vaccines have also been developed against bacteria. However, vaccines that are effective against viral diseases or parasitic diseases have been developed only for vaccines against a part of salmonid fishes and the iridovirus of the perch (Patent Document 1).

  Vaccines are generally used for the prevention or treatment of viral infections. Vaccines include inactivated vaccines (Japanese encephalitis, Weil disease, etc.), toxoids (tetanus, diphtheria, etc.), attenuated vaccines (BCG, polio, etc.), and genetically modified vaccines (eg, hepatitis B virus). Inactivated vaccines and toxoids detoxified with exotoxins are relatively safe vaccines that induce antibodies against them. Genetically modified vaccines are considered safer because they do not contain impurities when compared to inactivated vaccines.

  However, antibody production can be induced in these vaccines, but cellular immunity is less likely to be induced. Further, inactivated vaccines and attenuated vaccines need to obtain a large amount of virus as an antigen industrially, and it is essential to secure appropriate cells. Furthermore, the immune effect obtained with the attenuated vaccine is often maintained for a long period of time, but side effects and risks have been pointed out. Inactivated vaccines and genetically modified vaccines are considered to have a short antigen persistence in the host, and require an adjuvant or the like. Since these conventional vaccines need to be refrigerated during the period from production to inoculation of a subject, there are problems in that cost increases and efficacy decreases.

  Recently, research and development of vaccines has progressed, and a new vaccine species (DNA vaccine) that induces immunity has been developed by administering plasmid DNA encoding an immunogenic protein. Profits are improving. In other words, DNA vaccines can induce not only humoral immune responses but also cellular immunity, so that they can provide protection against infections and can be highly purified, at room temperature or at high temperatures. However, it is stable, refrigerated storage is not essential, it can be stored for a long time, it is easy to quickly improve DNA vaccines by genetic engineering techniques, and there are advantages such as shortening the time spent on vaccine development .

It is known that the immune response of rainbow trout is stimulated by injecting into the muscle a gene encoding glycoprotein, a constituent protein of Rhabdovirus (Non-patent Document 5). There is also a report on DNA vaccines for rainbow trout (Non-patent Document 6). However, there are no reports of DNA vaccines in other fish species.
Moreover, there is no report of a vaccine for stimulating protective immunity against flounder viral hemorrhagic sepsis virus infection.

Japanese Patent Laid-Open No. 9-176043 Takano et al., “Bulletin of the European Association of Fish Pathology” (Scotland), 2000, 20, 5, p. 186 Muroga Kiyokuni, Shuzo Egusa, “Introduction to Fish Diseases”, (Hoshiseisha Koseiku), 1996, p. 34 Isshiki and Nagano Taizo, 1. About the viral hemorrhagic sepsis of Japanese flounder, "Summary of the 7th Fisheries Research Presentation", held on March 12, 2002 Nishizawa et al., “Disees of Aquatic Organisms” (Germany), 2002, 48, p. 143-148 P. Boudinott et al., “Virology” (USA), 1998, 249, p. 297-306 McLauchlan et al., “Fish and shellfish Immunology” (UK), 2003, 15, p. 39-50

  It is an object of the present invention to provide a fish DNA vaccine for inducing protective immunity against flounder viral hemorrhagic sepsis.

As a result of intensive research on an effective vaccine against flounder viral hemorrhagic sepsis, the present inventors inoculated flounder with a plasmid DNA having a gene encoding a glycoprotein of flounder viral hemorrhagic septic virus (VHSV). However, the present invention was completed by finding that it has an immune effect against flounder viral hemorrhagic sepsis and that the expression level of immune-related genes increases. That is, the present invention
1. A DNA vaccine for fish, comprising as an active ingredient DNA comprising a nucleotide sequence encoding an immunogenic polypeptide against flounder viral hemorrhagic sepsis virus, or an expression vector comprising said DNA,
2. The DNA vaccine for fish according to the above 1, wherein the immunogenic polypeptide is a glycoprotein of flounder virus hemorrhagic septic virus or a partial fragment thereof,
3. The DNA vaccine for fish according to 2 above, wherein the glycoprotein is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof,
4). The immunogenic polypeptide is (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, and (2) one or more amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2. Or a modified polypeptide comprising an added amino acid sequence and having immunogenicity against the Japanese flounder virus hemorrhagic sepsis virus, or (3) 80% homology with the amino acid sequence represented by SEQ ID NO: 2 The fish DNA vaccine according to 1 above, which is a homologous polypeptide having immunogenicity against flounder viral hemorrhagic sepsis virus, or a partial fragment thereof,
5). The nucleotide sequence is (1) a nucleotide sequence represented by SEQ ID NO: 1, or (2) a homology with the nucleotide sequence represented by SEQ ID NO: 1 is 80% or more, and the flounder viral bleeding The DNA vaccine for fish according to the above 1, which is a nucleotide sequence encoding a polypeptide having immunogenicity against sepsis virus, or a partial sequence thereof,
6). 2. The fish DNA vaccine according to 1 above, wherein the expression vector is the plasmid pCMV-VHSVg of accession number FERM P-19504,
7). A method for preventing or treating viral hemorrhagic sepsis, which comprises administering to a fish the DNA vaccine for fish according to any one of 1 to 6 above,
8). 8. The method according to 7 above, wherein the fish is a fish belonging to the flounder family or the flounder family, and 7. The use of the fish DNA vaccine according to any one of the above 1 to 6 for inducing an immune response against flounder viral hemorrhagic sepsis.

  According to the fish DNA vaccine of the present invention, it is possible to confer immunity against viral hemorrhagic sepsis caused by flounder VHSV. More specifically, according to the fish DNA vaccine of the present invention, an immune response (humoral immune response and cellular immune response against flounder VHSV infection or viral hemorrhagic sepsis caused by flounder VHSV infection). Therefore, it is effective for the prevention or treatment of Japanese flounder VHSV infection or the prevention or treatment of the viral hemorrhagic sepsis. For example, pCMV-VHSVg that can be used as an active ingredient of the fish DNA vaccine of the present invention is a result of confirming a significant increase in the expression level of viral hemorrhagic sepsis virus infection test in flounder and immune-related genes. Therefore, it is effective as an active ingredient of a DNA vaccine, and can be expected to prevent the infection of flounder with viral hemorrhagic sepsis virus.

The fish DNA vaccine of the present invention is not particularly limited as long as it is a DNA construct comprising at least one nucleotide sequence encoding an immunogenic polypeptide against flounder viral hemorrhagic sepsis virus. In the fish DNA vaccine of the invention, for example,
(A) DNA containing a nucleotide sequence encoding an immunogenic polypeptide against flounder viral hemorrhagic sepsis virus, or (b) an expression vector containing said DNA (a). The DNA (a) can further include various regulatory sequences necessary for expression of the immunogenic polypeptide, and the expression vector (b) can also include the regulatory sequences.
As used herein, “immunogenic polypeptide against flounder viral hemorrhagic sepsis virus” means a polypeptide capable of inducing immunity against flounder VHSV (including humoral immunity and cellular immunity) in vivo. means.

  The immunogenic polypeptide against flounder VHSV is not particularly limited as long as it is a polypeptide capable of inducing immunity against flounder VHSV (including humoral immunity and cellular immunity) in vivo. For example, five proteins that are structural proteins of viruses belonging to the Rhabdoviridae family (especially flounder VHSV) (Kurokuni Muraga, Shuzo Egusa, Introduction to Fish Diseases): glycoprotein, nucleocapsid protein, RNA-dependent Mention may be made of RNA synthetases, and matrix proteins M1 and M2 (Kurokuni Muraga, Shuzo Egusa, Introduction to Fish Diseases), and partial fragments thereof. The immunogenic polypeptide is preferably a flounder VHSV glycoprotein or a partial fragment thereof, more preferably a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof.

  The immunogenic polypeptide further includes (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, and (2) one or more amino acids in the amino acid sequence represented by SEQ ID NO: 2. A modified polypeptide having an amino acid sequence deleted, substituted, or added, and having immunogenicity against flounder viral hemorrhagic sepsis virus, or (3) the amino acid sequence represented by SEQ ID NO: 2 Homology is 80% or more (preferably 85% or more, more preferably 90% or more, more preferably 95% or more, most preferably 98% or more), and the immunogen against flounder viral hemorrhagic sepsis virus The homologous polypeptide which has sex, or those partial fragments can be mentioned.

  In the present specification, the modified polypeptide is one or more (for example, 1 to several, preferably 1 to 10, more preferably 1 to 5, more preferably 1) in the amino acid sequence represented by SEQ ID NO: 2. 1 to 3, more preferably 1 to 2, particularly preferably 1 amino acid modification (eg, deletion, substitution, and / or addition), and the present invention still applies Means that can immunize fish.

  In this specification, homology in amino acid sequences means that two types of amino acid sequences are compared and analyzed by computer analysis software (SDC software), and two types of amino acids are present when the same type of amino acid is present at the same position. Means the identity calculated as being the same in the sequence.

  Examples of the nucleotide sequence encoding the immunogenic polypeptide used in the present invention include nucleotide sequences encoding each of the immunogenic polypeptides listed so far, for example, viruses belonging to the Rhabdoviridae family (particularly, , Flounder VHSV), a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, the modified polypeptide, the homologous polypeptide, or a nucleotide sequence encoding a partial fragment thereof.

  As the nucleotide sequence, (1) the nucleotide sequence represented by SEQ ID NO: 1 or (2) the homology with the nucleotide sequence represented by SEQ ID NO: 1 is 80% or more, and the flounder viral bleeding A nucleotide sequence encoding a polypeptide having immunogenicity against septic virus, or a partial sequence thereof is preferred. In this specification, homology in nucleotide sequence means that two types of nucleotide sequences are compared and analyzed by computer analysis software (SDC software), and two types of nucleotides are present when the same type of nucleotide is present at the same position. Means a value calculated as being identical in the array. The length of the partial sequence is particularly limited as long as the polypeptide encoded by the partial nucleotide sequence can induce immunity against flounder VHSV (including humoral immunity and cellular immunity) in vivo. Is not to be done.

  In addition, the nucleotide sequence encoding the immunogenic polypeptide may be naturally derived or totally synthesized, or synthesized using a part of the naturally derived one. But you can.

  A nucleotide sequence encoding an immunogenic polypeptide (for example, glycoprotein) used in the present invention can be obtained, for example, from a virus belonging to the rhabdoviridae family, specifically a Japanese flounder virus hemorrhagic septicemia virus. A typical method for obtaining the nucleotide sequence used in the present invention is a method commonly used in the field of genetic engineering, for example, screening using an appropriate DNA probe prepared on the basis of partial amino acid sequence information. The method of performing etc. is mentioned.

  The expression vector used in the present invention is not particularly limited as long as it is a vector that can be expressed in fish cells. The expression vector used in the present invention can be constructed on the basis of a self-replicating vector, that is, as an extrachromosomal independent substance, and its replication does not depend on chromosomal replication, for example, a plasmid. In addition, the expression vector may be incorporated into the genome of the host when it is introduced into the host and replicated together with the chromosome into which it has been integrated. As a procedure and method for constructing an expression vector that can be used in the present invention, those commonly used in the field of genetic engineering can be used.

  Examples of transcriptional regulatory sequences that can be used in the present invention include constitutive promoters, inducible or regulatory promoters, tissue-specific promoters, promoters derived from the genes of expressed antigens, and the like. As long as it can be expressed in the cell, it is not particularly limited thereto. Examples of the constitutive promoter include a promoter sequence derived from cytomegalovirus (CMV), or rous sarcoma virus (RSV), simian virus-40 (SV-40), muscle β-actin promoter, or herpes simplex virus (HSV). Examples include strong promoters. Examples of the tissue-specific promoter include a thymidine kinase promoter. Inducible or regulatable promoters include, for example, growth hormone regulatable promoters, promoters under the control of the lac operon sequence, or zinc inducible metallothionein promoters. The transcriptional regulatory sequence can be operably linked to a nucleotide sequence encoding an immunogenic polypeptide (ie, so that expression of the nucleotide sequence can be regulated).

  The regulatory sequence can include an expression control sequence including a promoter (eg, the inducible or constitutive promoter) DNA sequence, optionally further comprising an enhancer element, transcription or polyadenylation signal [eg, simian virus-40. One or more copies of the intron sequence for splicing (derived from (SV-40) or bovine growth hormone), or the immunostimulatory DNA sequence known as the CpG motif may be included.

  In addition, the expression vector may be selected, for example, from a bacterial replication origin sequence, or an antibiotic resistance (eg, kanamycin) gene or a non-antibiotic resistance gene (eg, β-galactosidase gene) for selection. Sex markers can be included.

Oligonucleotides with unmethylated CpG nucleotides are known to activate the immune system (A. Krieg et al ., Nature, 1995, 374, 546-549). Depending on the flanking sequence, certain CpG motifs are more immunostimulatory to B cell or T cell responses and preferentially stimulate certain species. The copy of the CpG motif in the DNA expression vector acts as an adjuvant that elicits an immune response against the expressed protein. A DNA extension comprising a CpG motif, ie, a CpG dinucleotide within the specified sequence, can be selected from about 5-40 base pairs in length. Multiple CpG motifs may be inserted into non-coding regions of the expression vector. When a humoral response is desired, a preferred CpG motif is a CpG motif that stimulates secretion of cytokines known to stimulate CD8 + T cell responses.

  The fish to which the present invention can be applied is not particularly limited as long as it is a fish that can be infected by flounder VHSV. For example, fish species belonging to the flounder family or the flounder family (for example, flounder) Or flounder etc.) or sweetfish or black sea bream.

  Examples of the DNA vaccine inoculation method include oral administration, intramuscular injection, intraperitoneal injection, administration using a gene gun, and immersion, and intramuscular injection and administration using a gene gun are preferable. Administration using a gene gun is a method in which a plasmid is coated with gold particles of about 1 μm in size, and high-pressure helium gas is used to shoot the skin, cells, or tissues of the subject with a special instrument in the manner of an air gun. is there. Compared with intramuscular injection, this gene gun can provide the same immune effect with 100 to 1000 times less DNA, and it has better reproducibility than intramuscular injection. It has excellent characteristics.

  Adjuvants stimulate the immune system and enhance the immune response to antigens, and are mainly added as adjuvants to vaccines. As typical adjuvants, for example, aluminum compounds, polynucleotides or bacterial cell components are known, but there are many of these that do not provide sufficient effects for application to the present invention. . In particular, since the action of an adjuvant is widely effective for antigenic substances, there is a risk of enhancing the antigenic stimulation of impurities contained in the antigen and causing harmful side effects, and it is necessary to give due consideration to the purity of the antigen used. There are problems such as. Among them, for example, IL-1β has been reported to be effective as an adjuvant (J. Y. Scheerlinck, Genetic adjuvants for DNA Vaccine, 19, 2647-2656, 2001). In the present invention, a plasmid into which IL-1β gene has been inserted so that it can be expressed in the body of fish (eg, flounder) can be prepared and inoculated into fish together with the vaccine of the present invention.

  The immune mechanism exerts a variety of physiological functions while cells that play various roles mutually regulate the function. T cell antigen receptor (TCR), major histocompatibility complex (MHC), or immunoglobulin (Ig) present on the cell surface of T cells and B cells, factors that play an important role in host defense It is possible to examine the activation of the immune system using the expression level of In the present invention, for example, in order to analyze the activation of the biological defense mechanism in the fish after inoculating the flounder with a DNA vaccine against flounder viral hemorrhagic sepsis, for example, changes in the expression level of TCR, MHC, and Ig Real-time PCR can be quantitatively confirmed for the activation of the immune system.

  Real-time PCR is a detection technique for profiling the PCR reaction curve by tracking the gene amplification process by PCR in real time using a fluorescence detector. When the sample to be examined is amplified by PCR, it is possible to calculate an accurate DNA amount by examining the number of PCR cycles that reach the exponential increase curve. For real-time PCR, for example, TaqMan manufactured by Perkin-Elmer or iCycler manufactured by BioRad can be used.

  In real-time PCR, two types of primers for real-time PCR (SG) and standard DNA (SG200) are used. For real-time PCR (SG), the amplicon should be designed to be short (60-100 bp) and low GC content. Generating a primer in the 3 ′ UTR portion makes it possible to design a gene-specific primer relatively easily and accurately. There is also a method of designing on a computer using software (for example, primer express; PE Biosystems Japan). A standard DNA primer (SG200) is designed outside the primer designed by real-time PCR. If you want to compare genes, it is desirable to have amplicons.

For preparation of standard DNA, first use a primer (SG200) specific to each gene to be measured, and react a total volume of 50 μL of PCR until it reaches a plateau (PCR is treated at 95 ° C. for 2 minutes, then at 95 ° C. for 30 minutes For 30 seconds at 55 ° C for 30 seconds and 72 ° C for 1 minute). Thereafter, the PCR product is removed using a column (for example, Microcon-PCR Centrifugal Filter Devices; manufactured by MILLIPORE), the excess primer is removed, and the concentration is measured. The copy number is calculated from the Avogadro constant (1 mol = 6.022 × 10 ²³ molecules). Next, the PCR product makes a 21-step dilution series (copy number 10 ¹³ to 10 ⁻⁷ ), and repeats PCR using the primer (SG) designed on the inside. At this time, the number of cycles is 14 so that amplification occurs exponentially. The PCR product thus obtained is subjected to agarose gel electrophoresis, and five steps after the band is completely invisible are amplified exponentially. Therefore, these five steps are used as standard DNA for actual real-time PCR.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely in detail, this invention is not limited to these.

Example 1: Isolation of a glycoprotein gene derived from Japanese flounder viral hemorrhagic sepsis virus (VHSV) (1) Preparation of PCR primers
Corresponding DNA based on the amino acid sequence of the rainbow trout viral hemorrhagic sepsis virus glycoprotein gene (GenBank = AF345857) reported by JR Winton and K. Einer-Jensen or P. Ahrens and K. Einer-Jensen From the base sequence, the following PCR primers were prepared according to a conventional method.
VHSV-glys: 5-TTTGCTAGCATGGAATGGAATACTTTTTTCTTG-3 (SEQ ID NO: 3)
VHSV-glya: 5-TTTGAATTCTCAGACCATCTGACTTCTGG-3 (SEQ ID NO: 4)

(2) Isolation of cDNA Add fetal bovine serum (FBS) to a commercially available L-15 medium (Invitrogen Corporation) to 20%, and the final concentrations of penicillin and streptomycin are 100 units / mL and 100 μg / mL, respectively. EPC cells [Fijian, ND Sulimanavois et al., Some properties of the epithelioma papulosum cyprini (EPC) cell line from carp Cyprinus carpio . Annual Viology, 134E, 207-220, 1983 ] Was cultured at 20 ° C. EPC cells cultured to confluence were infected with flounder viral hemorrhagic sepsis virus krrv 9822 strain (Nishizawa et al . Dis. Aquat. Org. 2002.48.143-148) stored at -80 ° C. It was. CPE (Cytopathic Effect) can be observed 2 to 7 days after infection of EPC cells with krrv 9822 strain.

150 μL of krrv 9822 strain culture solution (1.0 × 10 ⁸ TCID ₅₀ / mL) and 1 mL of TRIzol (GIBCO) were mixed and allowed to stand at room temperature for 5 minutes. Next, 200 μL of chloroform was added and mixed well, allowed to stand at room temperature for 5 minutes, then centrifuged (15,000 rpm, 4 ° C., 5 minutes), and the upper layer was transferred to a new centrifuge tube. An equal amount of isopropyl alcohol was added to the upper layer and left at room temperature for 10 minutes. After centrifugation (15,000 rpm, 10 minutes), the upper layer was discarded and the pelleted genomic RNA was dried and dissolved in 10 μL of diethylpyrocarbonate (DEPC) -treated water.

From thus extracted genomic RNA, commercially available cDNA synthesis kit; by ^{(cDNA SYNTHESIS KIT 1 st STRAND with} AMV Reverse Transcriptase Takara Shuzo Co., Ltd.), cDNA was synthesized according to the attached method of operation. Using this cDNA as a template, PCR was performed according to a conventional method using the PCR primer prepared in Example 1 (1). PCR included 10xbuffer 5μL, dNTP 4μL, VHSV-glys 1μL (25 pmol/μL), VHSV-glya 1μL (25 pmol/μL), DNA polymerase 0.5μL (5units / μL), cDNA 3μL (10ng), and sterile water 35.5μL to 50μL, treated at 95 ° C for 5 minutes, then treated 30 times at 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 2 minutes, then 72 ° C For 2 minutes. As a result of agarose gel electrophoresis of the obtained PCR product, it was visually confirmed that a DNA fragment of about 1.5 kbp was amplified.

(3) Analysis of DNA base sequence The amplified DNA fragment was inserted into a pGEM-T Eazy vector (Promega), a fluorescently labeled M13 primer (Nisshinbo Co., Ltd.) and a commercially available sequence kit (Thermo Sequence). Fluorescent Labeled Primer Cycle Sequencing Kit with 7-deaza-dGTP (manufactured by amersham pharmacia bitech) was used to prepare a sample by the dideoxy method and then a DNA sequencer (DNA sequencer model 4000; manufactured by LI-COR) Was used to determine the base sequence. When this sequence was analyzed, the DNA fragment amplified by PCR had an open reading frame (ORF) consisting of 1524 bp (SEQ ID NO: 1). The protein predicted from this ORF is 507 amino acid residues, and as a result of homology search (FASTA), reported by JR Winton and K. Einer-Jensen or P. Ahrens and K. Einer-Jensen The homology of the rainbow trout viral hemorrhagic septic virus glycoprotein gene (GenBank = AF345857) with the DNA base sequence was 85.6%, and the homology with the amino acid sequence was 91.5%. In addition, the homology with the DNA sequence of the glycoprotein gene (GenBank = AY167587) of Japanese flounder viral hemorrhagic septic virus reported by SM Kim and SI Park is 99.0%, and the homology with the amino acid sequence is It was 98.6%. Hereinafter, the resulting glycoprotein of Japanese flounder virus hemorrhagic septic virus may be referred to as “VHSVg”.

Example 2: Construction of plasmid pCMV-VHSVg The plasmid used in the analysis of the DNA base sequence of Example 1 (3) was treated with Eco RI and Nhe I. The glycoprotein gene derived from the krrv 9822 strain obtained in Example 1 (2) excised from the pGEM-T Easy vector is replaced with the human cytomegalovirus-derived promoter region of the pCDNA3.1 vector (Invitrogen). The plasmid pCMV-VHSVg was constructed by inserting into the Eco RI and Nhe I recognition sites of the multicloning site located downstream of the encoded sequence. In addition, pCMV-VHSVg is the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center on August 29, 2003 (Address: 1 1 Higashi 1-chome, Tsukuba City, Ibaraki, Japan 305-8566, Japan No. 6) was deposited domestically and its deposit number is FERM P-19504.

Example 3: Confirmation of glycoprotein gene expression
CCM cells (RIKEN BioResource Center) were transfected with pCMV-VHSVg using a commercially available transfection reagent (Effectene ^™ Transfection Reagent; manufactured by QIAGEN), and then cultured for 48 hours. Next, the cultured cells are collected in a centrifuge tube, centrifuged (3,000 rpm, 5 minutes), the medium alone is discarded, and the total RNA of the cultured cells is extracted in the same manner as in Example 1 (2). PCR was performed. As a result, it was confirmed that the glycoprotein gene was expressed. Furthermore, the molecular weight of VHSVg was confirmed using a commercially available transcription translation system (TnT Quick Coupled Transcription / Translation system; promega).

FIG. 1 shows the result of confirming the expression of the glycoprotein gene, and FIG. 2 shows the result of confirming the molecular weight of VHSVg.
Each lane in FIG. 1 is as follows:
Lane 1: Molecular size marker obtained by treating λ DNA with restriction enzyme Hind III Lane 2: COS cell transformed with pCMV-VHSVg Lane 3: COS cell transformed with pCDNA vector (control)
Lane 4: untreated COS cells Lane 5: 100 bp DNA marker Each lane in FIG. 2 is as follows:
Lane M: Wide molecular size marker Lane 1: pCMV-VHSVg
Lane 2: pCDNA3.1 vector

Example 4: Introduction of pCMV-VHSVg into flounder 10.0 μg of pCMV-VHSVg per flounder together with 50 μL of phosphate buffered saline (PBS) and into a muscle using a syringe equipped with a 27G needle Vaccinated.

Example 5: Infection protection test against flounder In each test section, flounder larvae (total length: about 5 cm, average fish weight: 2 g) were used. The test fish were bred in a 60 L water tank by circulating natural filtered seawater and bred at an average water temperature of 13.0 ° C.
First, according to the method of Example 4, pCMV-VHSVg 10.0 μg, PBS 50 μL as a control, pCDNA3.1 vector 10.0 μg, or VHSV (1.0 × 10 ² TCID ₅₀ / fish) inactivated by formalin were each transferred to flounder. Vaccinated. 30 days after the inoculation, the culture supernatant of krrv9822 strain cultured in EPC cells (L-15 medium + 20% FBS) in the same manner as in Example 1 (2) was added at 1.0 × 10 ² TCID _50/50 μL or 1.0 ₅₀ μL per flounder was inoculated by intraperitoneal injection at a concentration diluted to × 10 ³ TCID _50/50 μL. The compartments where the infection protection test was conducted are as follows.
Test group 1: pCMV-VHSVg + 1.0 × 10 2 TCID 50 krrv 9822 strain / flounder 1 fish test group 2: pCDNA3.1 vector +1.0 × 10 ² TCID ₅₀ krrv 9822 strain / flounder 1 fish test group 3: PBS + 1. 0 × 10 ² TCID ₅₀ krrv 9822 strain / flatfish 1 tail test area 4: formalin inactivated VHSV + 1.0 × 10 ² TCID ₅₀ krrv 9822 strain / flounder 1 tail test area 5: pCMV−VHSVg + 1.0 × 10 ³ TCID ₅₀ krrv 9822 strain / flounder 1 fish test group 6: pCDNA3.1 vector +1.0 × 10 ³ TCID ₅₀ krrv 9822 strain / flounder 1 fish test group ^{7: PBS + 1.0 × 10 3} TCID 50 krrv 9822 strain / flounder 1 fish test group 8: Formalin-inactivated VHSV + 5 × 10 ³ TCID ₅₀ krrv 9822 strain / flounder 1

Continue to observe flounder for 2 weeks after infection with krrv 9822 strain, and calculate cumulative mortality (number of dead / number of tested individuals) x 100 (%) of flounder in test section 1 to test section 8 and calculate the result as 1.0 × 3 and 4 and Table 1 are divided into 10 ² TCID _50/50 μL-infected group (test group 1 to 4) and 1.0 × 10 ³ TCID _50/50 μL-infected group (test group 5 to 8). “RPS” in the table is an abbreviation for relative percent survival, and is calculated by the following formula:
RPS = {1- (X / C)} × 100
[In the formula, the symbol X is “% mortality in the vaccination group” and C is “mortality in the% negative control group”]
The effectiveness of the vaccine was determined by comparison.
As a result, test plots 2, 3, and 4 had a cumulative mortality rate of about 96, 73, and 91%, respectively, while that of test plot 1 was about 5%. In addition, since test plots 6, 7, and 8 had a cumulative mortality rate of approximately 95, 100, and 90%, respectively, that of test plot 5 was approximately 0%, indicating that pCMV against krrv 9822 strain infection -Effectiveness of infection protection of VHSVg, that is, vaccine effect became clear.

  The DNA vaccine of the present invention can be applied for use in the prevention or treatment of Japanese flounder viral hemorrhagic sepsis.

   The numerical heading <223> in the sequence listing below describes the “Artificial Sequence”. Specifically, the base sequence represented by SEQ ID NO: 3 is primer VHSV-glys, and the base sequence represented by SEQ ID NO: 4 is primer VHSV-glya.

It is a photograph replacing a drawing showing the expression of a glycoprotein gene from COS cells after 48 hours of transfection with pCMV-VHSg. It is the photograph which replaces drawing which shows the molecular weight of VHSVg using a commercially available transcription translation system (TnT Quick Coupled Transcription / Translation system; promega). It is a graph which shows the time-dependent change of the cumulative mortality of Japanese flounder by DNA vaccine treatment in the 1.0 × 10 ² TCID _50/50 μL infection group regarding the cumulative mortality after VHSV (1.0 × 10 ² TCID ₅₀ / fish) challenge. It is a graph which shows the time-dependent change of the cumulative mortality of Japanese flounder by DNA vaccine treatment in the 1.0 × 10 ³ TCID _50/50 μL infection group regarding the cumulative mortality after VHSV (1.0 × 10 ³ TCID ₅₀ / fish) challenge.

Claims (9)

  A DNA vaccine for fish comprising, as an active ingredient, DNA comprising a nucleotide sequence encoding an immunogenic polypeptide against flounder viral hemorrhagic sepsis virus, or an expression vector comprising said DNA.   The fish DNA vaccine according to claim 1, wherein the immunogenic polypeptide is a glycoprotein of flounder virus hemorrhagic septic virus or a partial fragment thereof.   The fish DNA vaccine according to claim 2, wherein the glycoprotein is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 2 or a partial fragment thereof.   The immunogenic polypeptide is (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2, and (2) one or more amino acids are deleted or substituted in the amino acid sequence represented by SEQ ID NO: 2. Or a modified polypeptide comprising an added amino acid sequence and having immunogenicity against flounder viral hemorrhagic sepsis virus, or (3) 80% homology with the amino acid sequence represented by SEQ ID NO: 2 The fish DNA vaccine according to claim 1, which is a homologous polypeptide having immunogenicity against flounder virus hemorrhagic septic virus, or a partial fragment thereof.   The nucleotide sequence is (1) a nucleotide sequence represented by SEQ ID NO: 1, or (2) a homology with the nucleotide sequence represented by SEQ ID NO: 1 is 80% or more, and further, a flounder viral hemorrhagic property The DNA vaccine for fish according to claim 1, which is a nucleotide sequence encoding a polypeptide having immunogenicity against sepsis virus, or a partial sequence thereof.   The fish DNA vaccine according to claim 1, wherein the expression vector is a plasmid pCMV-VHSVg of the accession number FERM P-19504.   A method for preventing or treating viral hemorrhagic sepsis, comprising administering the fish DNA vaccine according to any one of claims 1 to 6 to a fish.   The method according to claim 7, wherein the fish is a fish belonging to the flatfish family or the flatfish family.   Use of the fish DNA vaccine according to any one of claims 1 to 6 for inducing an immune response against flounder viral hemorrhagic sepsis.

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