WO2013165190A1 - Vaccine for preventing viral hemorrhagic sepsis of flatfishes - Google Patents
Vaccine for preventing viral hemorrhagic sepsis of flatfishes Download PDFInfo
- Publication number
- WO2013165190A1 WO2013165190A1 PCT/KR2013/003811 KR2013003811W WO2013165190A1 WO 2013165190 A1 WO2013165190 A1 WO 2013165190A1 KR 2013003811 W KR2013003811 W KR 2013003811W WO 2013165190 A1 WO2013165190 A1 WO 2013165190A1
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- WIPO (PCT)
- Prior art keywords
- vaccine
- virus
- flounder
- viral hemorrhagic
- vhs2010
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
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- C—CHEMISTRY; METALLURGY
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Definitions
- the present invention relates to a vaccine for preventing viral hemorrhagic sepsis of olive flounder.
- Viral Hemorrhagic Septicemia is a disease that causes massive mortality in freshwater and seawater fish worldwide. In Korea, since it was first reported in halibut in 2001, it has been consistently confirmed in Gyeongbuk and Jeju, and in the late autumn and spring when the water temperature decreases, it causes death in not only small fry but also big fish, which causes great loss to the aquaculture industry. have. Viral hemorrhagic sepsis is caused by infection with the viral hemorrhagic sepsis virus (VHSV), and fish such as flounder have blackened body color, abdominal distension and hernia, and die a week as early as a month later. Due to the tendency to pay attention, careful attention is required for the management of farms.
- VHSV viral hemorrhagic sepsis virus
- the causative agent of viral hemorrhagic sepsis is a single-stranded RNA virus that has the outer membrane of the genus Rhabdoviridae and Novirhabdovirus.
- the size of the virus particles is 50-70 ⁇ 180-240 nm, and the model of the virus particles is a bullet, a typical form of Rabdovirus, rounded on one side and flat on the other.
- Academia divides genotypes based on the glycoprotein gene sequences of VHSV isolated from fresh water, and these genotypes are American type (Genogroup I) and British Isles type (Genogroup II) depending on geographic separation. The three genotypic divisions of the European type (Genogroup III) are common.
- Genotypes isolated from Korea are found to be similar to viruses isolated from North America and Japan belonging to Genotype I.
- VHSV isolated from flounder has genetic distinction from the virus isolated from freshwater fish, but not serologically.
- Viral hemorrhagic sepsis is a marine animal infectious disease under Article 2 of the Fisheries Animal Disease Control Act. It is a viral disease that causes flounder mortality at low temperatures below 15 ° C. The mortality rate is high and contagious. It is a disease that is managed by restricting movement.
- IPNV infectious pancreatic ulcer virus
- DNA vaccine for the prevention of infectious hematopoietic necrosis from Norvartis animal health, Apex-IHN, Japan
- IPNV infectious pancreatic ulcer virus
- few vaccines are commercially available to prevent diseases caused by fish virus, and one of the Irido virus inactivated vaccines imported from Japan is a commercial vaccine.
- the vaccine of viral hemorrhagic sepsis has been studied on the structure and antigenic proteins of hemorrhagic sepsis virus, and based on these basic studies, studies on inactivating vaccines, recombinant protein vaccines, attenuated vaccines, DNA vaccines, etc. have been conducted.
- no vaccine has been developed that has secured stability and efficacy. This shows that it is very difficult to search for a suitable candidate virus for the production of a fish virus vaccine, to prepare the vaccine effectively and to ensure stability.
- the present inventors completed the present invention by analyzing a viral hemorrhagic sepsis occurring in Korea, selecting a vaccine candidate, and developing an optimal vaccine for preventing viral hemorrhagic sepsis of the flounder using the candidate strain.
- Another object of the present invention is to provide a method for preventing viral hemorrhagic sepsis of olive flounder using the vaccine.
- the present invention provides an inactivated vaccine for the prevention of viral hemorrhagic sepsis of the olive flounder containing inactivated viral hemorrhagic sepsis virus FP-VHS2010-01 antigen (KCTC 12153BP).
- the present invention provides a method for preventing viral hemorrhagic sepsis of the flounder, comprising administering the vaccine to the flounder.
- the vaccine according to the present invention can effectively prevent viral hemorrhagic sepsis occurring in the flounder, it can be usefully used for the aquaculture industry of the flounder.
- 1 to 4 shows glycoprotein sequences of the VHS virus FP-VHS2010-01 isolated in Example 1, NCBI AY167587 (NBCI), FP-VHS2005-01 (separated from Jeju, 2005), FP-VHS2010, which are known VHS viruses. This is compared with the glycoprotein sequences of -02 (2010 Pohang separation) and FP-VHS2011-01 (Jeju separation 2011).
- Figure 5 is a graph showing the cumulative mortality (%) after inoculation of the flounder of various sizes at various concentrations of the VHS virus FP-VHS2010-01 isolated in Example 1.
- Figure 6 shows the result of confirming the expression rate of immune genes in kidney tissue after inoculation on the flounder with the VHS virus FP-VHS2010-01 isolated in Example 1.
- a denotes a condition tree
- b denotes a scatter plot
- c denotes a scanned image.
- Figure 7 is a photograph of the structural protein of VHS virus FP-VHS2010-01 isolated in Example 1 by (a) SDS-PAGE and (b) Western blot using a polyclonal antibody.
- lane M is the marker and lane 1 is the VHS virus FP-VHS2010-01.
- Example 8 is a photograph of cells cultured at various temperatures after inoculation of the VHS virus FP-VHS2010-01 isolated in Example 1 into an EPC cell line.
- a represents normal cells
- b to d represent cells cultured at 15 ° C., 20 ° C., and 25 ° C. for 3 days after virus inoculation, respectively.
- FIG. 9 shows the results of incubating the VHS virus FP-VHS2010-01 isolated in Example 1 into the EPC cell line, followed by culturing in a culture flask (125 cm 2 ; a) and a roller culture bottle (850 cm 2 ; b), respectively. .
- Figure 11 is a photograph showing a change in the expression rate of immune-related genes according to the breeding water temperature of the flounder during the vaccine treatment method and vaccine treatment.
- a is a high temperature experimental group
- b is a low temperature experimental group
- c is a control group
- lanes 1 and 2 are vaccinated groups in a and b
- lanes 3 and 4 are vaccine injection groups
- lanes 3 and 4 are PBS administration group.
- FIG. 12 is a graph showing the survival rate of the flounder after the attack experiment according to the breeding water temperature of the flounder during the vaccine treatment method and vaccine treatment.
- Figure 13 is a graph showing the mean cumulative mortality (a), lysozyme activity (b), and ELISA results for VHSV when the challenge test after the vaccine treatment of the present invention.
- Figure 14 is a photograph of each organ tissue of the olive flounder after administration of the vaccine of the present invention by H & E staining.
- the present invention provides an inactivated vaccine for preventing viral hemorrhagic sepsis of olive flounder, which comprises the inactivated viral hemorrhagic sepsis virus FP-VHS2010-01 (KCTC 12153BP) antigen.
- Viral hemorrhagic sepsis (VHS) virus used in the vaccine of the present invention FP-VHS2010-01 is a strain isolated from the causative virus that caused the mass mortality of the flounder in Geoje farm, a known VHS virus (AY 167587 registered in GenBank) ; FP-VHS2005-01 isolated from Jeju in 2005; FP-VHS2010-02 isolated from Pohang in 2010; and FP-VHS2011-01 isolated from Jeju in 2011) (see FIGS. 1 to 4). ).
- VHS virus FP-VHS2010-01 When the strain used for the vaccine, VHS virus FP-VHS2010-01, is injected into the flounder at the concentration of 2 ⁇ 10 7 TCID 50 /, 2 ⁇ 10 6 TCID 50 / and 2 ⁇ 10 5 TCID 50 / At 6 days of injection, mortality of 50% or more is shown, and mortality of 90-100% is shown at 2 weeks of injection, and this mortality increases with smaller flounder and higher virus concentration.
- the VHS virus FP-VHS2010-01 increases the immune gene by 3 to 13 times or more when compared with the flounder uninfected. This shows that the VHS virus FP-VHS2010-01 is a highly pathogenic virus.
- Vaccines according to the invention can be prepared according to manufacturing methods commonly known in the art. The method includes culturing the vaccine strain, isolation of the virus strain, inactivation of the virus, and concentration and dilution.
- the VHSV vaccine can be obtained by culturing the VHS virus FP-VHS2010-01 at a temperature of 18 to 22 °C, preferably 20 °C as one of the optimal conditions. Such temperature conditions are advantageous for inducing maximal proliferation of the VHS virus FP-VHS2010-01 to augment the antigen in the vaccine of the present invention.
- an EPC cell line may be used, but is not limited thereto. Conventional cell lines known in the art, for example, BF-2, RTG-2, FHM, CHSE-214, and the like may be used.
- the vaccine of the present invention can be obtained by inactivating the cultured VHS virus with heat as one of the optimal conditions, preferably by leaving it for 2 to 5 days at a temperature of 50-70 ° C. Can be.
- the vaccine of the present invention may be prepared by inactivating the VHS virus using formarin, but formarin inactivation requires an additional process of removing used formarin and the inactivated virus is toxic in cell culture, It is preferable to deactivate using heat (see FIG. 10).
- the vaccine of the present invention is preferably inoculated by injection when the size of the flounder 13 ⁇ 17cm. Immersion is known as a vaccination method, but dipping shows lower efficiency than the injection method in terms of increased expression and mortality of immune-related genes.
- the vaccines of the present invention are advantageously inoculated at high temperatures (eg 18-22 ° C.) as compared to low temperatures (eg 10-14 ° C.) during treatment. The low temperature treatment shows lower efficiency than the high temperature treatment in terms of increased expression and mortality of immune-related genes.
- the vaccine of the present invention can be inoculated by high temperature / injection.
- Inactivated vaccines according to the invention may further comprise an adjuvant.
- the immunopotentiator may be used without limitation as long as it is known in the art.
- sugars, amino acids, mineral oils, vegetable oils, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, and the like may also be used.
- the vaccine according to the invention can be used by dispensing the vaccine solution in a vial of a suitable volume, for example about 10-500 ml volume, and then sealing.
- the present invention provides a method for preventing viral hemorrhagic sepsis of the flounder, comprising administering the vaccine to the flounder.
- the flounder includes fish such as flounder which are at risk of infection.
- the method for preventing viral hemorrhagic sepsis of the olive flounder using the inactivated vaccine of the present invention includes administering the vaccine of the present invention to the flounder using intraperitoneal administration or the like.
- VHSV Viral hemorrhagic sepsis virus
- VHS virus FP-VHS2010-01 selected in Example 1 was analyzed, and the known VHS virus (AY 167587 registered in GenBank; FP-VHS2005-01 isolated from Jeju in 2005; 2010 FP-VHS2010-02 isolated from Pohang and FP-VHS2011-01 isolated from Jeju in 2011).
- kidney and spleen tissues of the flounder infected with the VHS virus FP-VHS2010-01 were crushed with a homogenizer and total RNA was isolated using trizol. After cDNA was synthesized from the isolated total RNA, the cDNA was used as a template, and RT-PCR was performed using primers of SEQ ID NO: 1 and SEQ ID NO: 2.
- VHSG-exp-F 5'-GGATCCATGGAATGGAATACTTTTTCCTTGGTG-3 '(SEQ ID NO: 1);
- VHSG-exp-R 5'-AAGCTTGACCGTCTGACTTCTGGAGAA-3 '(SEQ ID NO: 2)
- RT-PCR contains 1 ⁇ PCR buffer (20 mM Tris-hydrochloride (pH 8.4), 50 mM KCl and 1.5 mM MgCl 2 ), 200 uM dNTP (Bioneer, Korea), 0.2 uM primer and 2U Taq DNA polymerase (Bioneer, Korea) and 50 ng of the template cDNA was mixed, followed by reaction at 94 ° C. for 5 minutes, 1 minute at 94 ° C., 1 minute at 55 ° C., and 1 minute at 72 ° C. for 30 minutes, followed by 7 minutes at 72 ° C. It was.
- the amplified PCR product was loaded on 1.5% agarose gel in 1X TAE (40 mM Tris-acetate, 1 mM EDTA) buffer, and then electrophoresed at 100 V for 15 minutes to confirm the size.
- the PCR product was separated from the gel and purified using QIAquick gel extraction kit (Qiagen, USA).
- the purified DNA was inserted into a pGEM-T easy vector (Promega, pGEM-T easy vector system, USA), and the plasmid was transformed by thermal shock to JM109 (Takara, Japan) competent cells.
- the transformed cells were plated in LB medium containing 40 ⁇ l of 2% (w / v) X-gal, 7 ⁇ l of 20% (w / v) IPTG solution, and 50 ⁇ g / ml of ampicillin and incubated at 37 ° C. for 18 hours. Then, white colonies into which the PCR product gene was inserted were selected. The colonies were incubated in LB medium (Yeasy extract 5 g / L, pancreatic digest of casein 10 g / L, sodium chloride 10 g / L, Difco; Miller) to which 50 ⁇ g / ml ampicillin (Sigma, USA) was added.
- LB medium Yeasy extract 5 g / L, pancreatic digest of casein 10 g / L, sodium chloride 10 g / L, Difco; Miller
- the glycoprotein of the VHS virus FP-VHS2010-01 was selected from the known VHS virus (AY 167587 registered with GenBank; FP-VHS2005-01 isolated from Jeju in 2005; FP-VHS2010-02 isolated from Pohang in 2010; And FP-VHS2011-01) isolated from Jeju in 2011 was confirmed to be a new strain showing a homology of 98 to 99%.
- an EPC cell line (ATCC No; CRL-2872) adapted to 20 ° C. was passaged in a 25 cm 2 culture flask, followed by TCID 12 hours later. Incubated by inoculating 200 ⁇ L of 10 7 / ml virus solution (medium 5 mL). Then, the characteristics were analyzed as follows.
- the cultured virus FP-VHS2010-01 was centrifuged and diluted to 10 8 TCID 50 / ml on 10 flounder so that the concentrations were 2 ⁇ 10 7 TCID 50 / head and 2 ⁇ 10 6 TCID 50 / head.
- the cumulative mortality was investigated for 2 weeks at 10-12 °C after artificial infection by intraperitoneal injection. As a result of the experiment, more than 50% of the flounder died on the 6th day of injection, and 90-100% of the flounder died on the 2nd week of injection.
- 20g group is 2 ⁇ 10 5 TCID 50 / mouse, and 1 ⁇ 10 4 TCID was injected at a concentration of 50 / mouse, 45g group is 4 ⁇ 10 5 TCID 50 / animal and 2.5 ⁇ 10 4 TCID injection at a concentration of 50 / mouse
- the 100g test group was injected at a concentration of 1 ⁇ 10 6 TCID 50 / head, and the 200g test group was injected at a concentration of 2 ⁇ 10 6 TCID 50 / head.
- the 20g experimental group (2 ⁇ 10 TCID 50 5 / mice) showed 50% mortality at 1 week and 80% mortality at 2 weeks, and the 45g experimental group (4 ⁇ 10 5 TCID 50 / head) was 20 at 1 week.
- % Mortality and 60% mortality at 2 weeks 100g experimental group (1 ⁇ 10 6 TCID 50 per animal) showed 0% mortality at 1 week and 30% mortality at 2 weeks, and 200g experimental group (2 ⁇ 10 6) TCID 50 per animal) showed 0% mortality at 1 week and 14% mortality at 2 weeks (see FIG. 5).
- the results show that the smaller the size of the fish, the higher the pathogenicity of the virus.
- the expression patterns of the immune genes in the kidney tissues of the flounder infected with the virus were analyzed using DNA chip microarrays (self-made). Confirmed. Specifically, after inoculation of the VHS virus FP-VHS2010-01 into the abdominal cavity of a 10 cm flounder at 12 ° C., the expression levels of the immune genes of the kidney and the spleen were measured in the control group (the kidney and spleen of the flounder of normal findings). Expression level of immune genes).
- 7% polyethylene glycol (PGE-6000) and 2.3% NaCl were added to the supernatant of the mass cultured virus solution, and then incubated overnight with stirring at 4 ° C. Centrifuge the culture at 20,000 g for 40 minutes to obtain pellets, resuspend in small amount of TNE buffer (0.01 M Tris HCl, 0.1 M NaCl, 0.001 M RDTA) and then on a 15% sucrose cushion Virus pellets were condensed and centrifuged at 115,000 g at 4 ° C. for 1 hour to obtain virus pellets.
- TNE buffer 0.01 M Tris HCl, 0.1 M NaCl, 0.001 M RDTA
- SDS-PAGE analysis was performed for analysis of the antigenic protein of the FP-VHS2010-01 strain.
- the virus was pulverized by sonication for 10 minutes, then mixed 1: 1 with 2 ⁇ sample buffer (0.12M Tris-HCl, 4% SDS, 20% glycerol, 5 mM 2-mercaptoethanol) and cut off at 100 ° C. for 10 minutes. Ready. 15 ⁇ l of the sample prepared on a 12% SDS-polyacrylamide gel was loaded and electrophoresed at 100 V for 1 hour 20 minutes. The gel was electrophoresed and stained with dye (coomassie brilliant blue R-250) and bleached to observe.
- dye coomassie brilliant blue R-250
- Two rabbits stabilized for two weeks were injected with the virus solution of the present invention at week zero. At 4 weeks, 6 weeks and 8 weeks, the first to third boost injections were performed with the same virus solution. IgG was then purified using a Protein A column from 10 mL of rabbit serum. The antigen specific antibody was then purified using an affinity column to which the antigen was bound. The purified antibody was dialyzed and concentrated. To bind FITC to the purified antibody, 2 mg of antibody was dialyzed, then mixed with FITC and stirred for 2 hours. Dialysis was performed to remove unbound free FITC to obtain an FITC bound antibody.
- Example ⁇ 4-2> the SDS electrophoresis gel was placed on a nitrocellulose membrane paper (nitrocelluolse menbrane paper, NC paper, Amersham Biosciences, Germany) and transferred at 100 V for 1 hour, and the NC paper was twined ( Tween 20) was washed five times with PBS (T-PBS) containing, and then blocked with 1% BSA (Bovine Serum Albumin, Sigma) for 1 hour and washed five times with T-PBS.
- the VHSV antibody prepared in Example ⁇ 4-3> was diluted to 1 / 10,00 and washed 5 times with T-PBS after 2 hours of reaction.
- goat anti-rabbit immunoglobulin (Sigma) coupled with alkaline phosphate was used, diluted to 1 / 5,000, reacted for 1 hour, washed five times with T-PBS, and BICP / NBT. Observation was carried out by the addition of phosphatase substrate (Sigma).
- the SDS-PAGE results are shown in (a) of FIG. 7, and the Western blot results are shown in (b) of FIG. 7.
- Lane M in each figure means a marker
- lane 1 is the result of loading FP-VHS2010-01.
- the structural protein of the VHS virus FP-VHS2010-01 of the present invention shown in FIG. 7 (a) relates to a VHS virus (Kim Soo-mi, cultured flounder, viral hemorrhagic septicemia virus (VHSV) infection occurring in Paralichthys olivaceus). Study, the Department of Fish Pathology, graduate School, Pukyong National University, Ph.D., p.101) showed the same protein pattern and different protein patterns from other viral HIRRVs. In addition, the results of Figure 7 (b) it was confirmed that the FP-VHS2010-01 virus antigenic in the antigen-antibody reaction.
- VHS virus FP-VHS2010-01 was inoculated into EPC cell lines known to have the highest proliferation rate of marine fish-derived VHSV and incubated under various temperature conditions (15 ° C, 20 ° C and 25 ° C) for 3 days, and then cell proliferation was analyzed. .
- EPC cell lines were cultured monolayer in MEM medium (welgene) to which 1% antibiotic (Gibco) and 10% FBS (Gibco) were added, and then the cultured EPC cell lines were inoculated with VHS virus and adsorbed at room temperature for 30 minutes. .
- CPE cytopathic effect
- FIG. 8. 8 a is a control
- b to d are photographs of cell lines cultured at 15 ° C., 20 ° C. and 25 ° C. after virus inoculation, respectively.
- EPC cell lines showed the fastest growth at 25 ° C
- virus proliferation showed the highest cytopathic effect at 20 ° C. This shows that the virus cell growth temperature is different from the normal cell line culture temperature.
- the VHS virus selected in Example 1 was inactivated through a formalin inactivation method and a thermal inactivation method, which is a method commonly used for virus inactivation.
- Cells were cultured to compare stability.
- the formalin inactivation method is thawed frozen virus cultures by centrifugation (40,000 rpm, 4 °C, 10 minutes) to remove the cell components, the supernatant treated with formalin at a ratio of 1: 4,000 (0.4%).
- the reaction was carried out at 37 ° C. for 3 days.
- the heat inactivation method was performed by inactivating the supernatant from which cell components were removed at 60 ° C. for 3 days.
- the prepared vaccine was used while stored at 4 °C.
- FIG. 10 a is a photograph of cells cultured for 2 days by inoculating EPC cells after inactivating the virus of the present invention with formarin
- FIG. 10 b is inoculating EPC cells after inactivating the virus of the present invention with heat. It is a photograph of the cells cultured for 2 days.
- the virus inactivated by formalin was found to be cytotoxic due to formalin toxicity, which was confirmed to be a problem in safety, and also a further process of removing formalin (inactivated reactant by centrifugation (X50).
- the effect of the treatment method and treatment temperature of the vaccine on the immune capacity was analyzed as follows.
- the flounder (average weight 24.6g, 13.4cm) was divided into high-temperature experimental group bred at 20 ° C and low-temperature experimental group bred at 12 ° C, followed by injection vaccine (10 6 TCID 50 / head, intraperitoneal injection) and immersion vaccine (10 6 TCID 50 / horses, soaked for 3 minutes, carried out once), and was inoculated with VHS virus 20 animals for each experimental group 2 weeks after breeding at 12 °C. Expression and cumulative mortality of immune-related genes were investigated after the inoculation.
- FIG. 11 a shows a high temperature experimental group
- b a low temperature experimental group
- c represents a control group (PBS treatment group).
- lanes 1 and 2 in the a and b are immersion groups
- lanes 3 and 4 are the vaccine injection groups
- lanes 1 and 2 in the c are negative controls
- lanes 3 and 4 are the PBS administration groups.
- the results show that the expression level of the immune-related genes was increased in the experimental group vaccinated with the hot and cold treatment compared to the control group.
- the expression level of the immune-related genes in the experimental group using the high-temperature treated vaccine was higher than the expression level of the immune-related genes in the experimental group using the low-temperature treated vaccine, it was confirmed that the vaccine was treated with high temperature.
- the cumulative mortality results are shown in FIG. 12.
- the mortality of the control was 77%, while the hot / vaccine injection group was 17%, the hot / vaccine injection group was 71%, the cold / vaccine injection group was 45%, and the cold / vaccine immersion group was 50%.
- % Mortality was shown. From the above results, it can be seen that the treatment at a higher temperature than the low temperature is more effective and the scanning method is more effective than the dipping. The high survival / vaccine injection group showed the highest relative survival rate of 78%, which was the most effective.
- the vaccine was inoculated on 20 flounder (average body weight 41g, 17cm), respectively.
- the VHS virus was injected with 10 5 TCID 50 / mice (0.1 ml / horse), followed by mortality against the control group (PBS administration), lysozyme activity, and aggregated antibody titers against Edward bacteria by ELISA.
- Lysozyme activity and aggregated antibody titer were performed by the following method.
- Solution A tertiary distilled water
- Solution B tertiary distilled water
- Micrococcus lysodeikticus (Sigma no.M-3370) was dissolved in 1 ml of LY solution (hereinafter, referred to as bacterial solution).
- test serum 50 ⁇ l was repeatedly dispensed into three wells of a 96 well flat bottom plate, and 50 ⁇ l of the bacterial solution was dispensed thereto.
- the activity was measured by calculating the formula ⁇ (0 min absorbance value)-(15 min absorbance value) ⁇ ⁇ 10000.
- the serum to be tested was dispensed 25 ⁇ l into the wells of the first row only.
- FIG. 13A the mortality rate of the PBS control group was 90%, whereas the virus alone had a mortality rate of 20% and a relative survival rate of 77%.
- Figure 13b and 13c it was confirmed that lysozyme activity and ELISA showed a higher activity than the control.
- each organ fixed in 10% BNF was cut and re-fixed in the same fixation solution within 48 hours. After 24 hours, the resultant was washed with running water for 6-12 hours and dehydrated with alcohol (70-100%) by concentration. Thereafter, the mixture was cleared with xylene and infiltrated with paraffin to make a paraffin block, and then cut into small portions (4 um) using a microtome and then attached to the slide. The attached slides were dried at 50 ° C. overnight and then stained by H & E staining and observed under a microscope.
- FIG. 14 shows micrographs of liver, spleen, kidney, heart, stomach and gills after VHSV vaccine treatment. As shown in the figure, normal findings were observed in all organs when the VHSV vaccine was administered, indicating that the VHSV vaccine of the present invention did not affect the safety of the flounder.
- the vaccine according to the present invention can effectively prevent viral hemorrhagic sepsis occurring in the flounder, it can be usefully used for the aquaculture industry of the flounder.
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Abstract
The present invention relates to an inactivated vaccine for preventing viral hemorrhagic sepsis of flatfishes including inactivated viral hemorrhagic sepsis virus FP-VHS2010-01(KCTC 12153BP) antigen, which can effectively prevent viral hemorrhagic sepsis infecting flatfishes, and can thus be applied to the industry of cultivating flatfishes etc.
Description
본 발명은 넙치의 바이러스성 출혈성 패혈증 예방용 백신에 관한 것이다.The present invention relates to a vaccine for preventing viral hemorrhagic sepsis of olive flounder.
바이러스성 출혈성 패혈증(Viral Hemorrhagic Septicemia, VHS)은 아이트베드(Egtved)병이라 불리며, 전세계적으로 담수 및 해수 어류에 발생하여 대량 폐사를 유발하는 질병이다. 우리나라의 경우, 2001년 넙치에서 처음 보고된 이후로, 경북 및 제주 지역에서 꾸준히 확인되고 있으며, 수온이 낮아지는 늦가을부터 봄철에 걸쳐 작은 치어 뿐만 아니라 큰 어체에도 폐사를 일으켜 양식업계에 큰 손실을 끼치고 있다. 바이러스성 출혈성 패혈증은 바이러스성 출혈성 패혈증 바이러스(VHSV)의 감염에 의해 발생하며, 감염된 넙치 등의 어류는 몸 색깔이 검어지고 복부 팽만과 탈장 등의 증상을 보이며, 빠르면 1주일, 늦을 경우 한달 후에 폐사하는 경향을 보이고 있어 양식어장 관리에 세심한 주의가 요구되고 있다.Viral Hemorrhagic Septicemia (VHS), called Ettved, is a disease that causes massive mortality in freshwater and seawater fish worldwide. In Korea, since it was first reported in halibut in 2001, it has been consistently confirmed in Gyeongbuk and Jeju, and in the late autumn and spring when the water temperature decreases, it causes death in not only small fry but also big fish, which causes great loss to the aquaculture industry. have. Viral hemorrhagic sepsis is caused by infection with the viral hemorrhagic sepsis virus (VHSV), and fish such as flounder have blackened body color, abdominal distension and hernia, and die a week as early as a month later. Due to the tendency to pay attention, careful attention is required for the management of farms.
바이러스성 출혈성 패혈증(VHS)의 원인바이러스는 단일가닥의 RNA 바이러스로 라브도비리데(Rhabdoviridae)과 노비라브도바이러스(Novirhabdovirus)속의 외막을 가진 바이러스이다. 50~70×180~240 nm의 크기이며 바이러스 입자의 모형은 전형적인 라브도바이러스의 형태인 탄환형으로, 한쪽은 둥글며 다른 한쪽은 편평하다. 학계에서는 담수에서 분리된 VHSV의 당단백질 유전자 염기서열을 기초로 유전형으로 구분하고, 이들 유전형은 지리적 분리 유래에 따라 아메리카 타입(American type; Genogroup Ⅰ), 영국제도 타입(British Isles type; Genogroup Ⅱ), 유럽 타입(European type; Genogroup Ⅲ)의 3가지의 유전형적 구분이 일반화되어 있다. 중화항체 시험에 의거해 3개의 혈청형으로 나누고 있는데 이들 혈청형 사이에는 교차반응이 일어난다. 우리나라에서 분리된 유전형은 유전형(Genogroup) Ⅰ에 속하는 북미지역과 일본에서 분리되는 바이러스와 유사하다고 밝혀져 있다. 또한 넙치에서 분리된 VHSV는 담수어에서 분리된 그 바이러스와 유전학적인 구분은 있지만 혈청학적인 구분은 되지 않는다. The causative agent of viral hemorrhagic sepsis (VHS) is a single-stranded RNA virus that has the outer membrane of the genus Rhabdoviridae and Novirhabdovirus. The size of the virus particles is 50-70 × 180-240 nm, and the model of the virus particles is a bullet, a typical form of Rabdovirus, rounded on one side and flat on the other. Academia divides genotypes based on the glycoprotein gene sequences of VHSV isolated from fresh water, and these genotypes are American type (Genogroup I) and British Isles type (Genogroup II) depending on geographic separation. The three genotypic divisions of the European type (Genogroup III) are common. Based on the neutralizing antibody test, it is divided into three serotypes, and cross-reactions occur between these serotypes. Genotypes isolated from Korea are found to be similar to viruses isolated from North America and Japan belonging to Genotype I. In addition, VHSV isolated from flounder has genetic distinction from the virus isolated from freshwater fish, but not serologically.
바이러스성 출혈성 패혈증은 수산동물 질병 관리법 제 2조의 수산동물전염병으로 15℃이하의 저수온기에 넙치의 폐사를 일으키는 바이러스성질병으로 폐사율이 높고 전염성이 강하기 때문에 종묘에서 검출되는 경우에는 방류를 금지하고 다른 지역으로의 이동을 제한하여 관리하고 있는 질병이다.Viral hemorrhagic sepsis is a marine animal infectious disease under Article 2 of the Fisheries Animal Disease Control Act. It is a viral disease that causes flounder mortality at low temperatures below 15 ° C. The mortality rate is high and contagious. It is a disease that is managed by restricting movement.
바이러스성 출혈성 패혈증은 일단 발병하면 빠른 속도로 전염되기 때문에, 질병의 예방이 매우 중요하지만, 사육환경 및 사육관리의 개선만으로는 질병 발생을 억제하는데 한계가 있다. 따라서 보다 근본적인 예방 대책으로서 백신의 개발이 시급한 실정이다.Since viral hemorrhagic sepsis is transmitted rapidly at the onset of disease, prevention of disease is very important, but improvement of the breeding environment and breeding management alone has a limit in suppressing disease occurrence. Therefore, it is urgent to develop a vaccine as a more fundamental preventive measure.
현재까지 세균으로 인한 질병을 예방하기 위한 백신은 많이 개발되어 있으나, Pharmaq사의 전염성 췌장 궤양증 바이러스(IPNV)에 대한 백신과 Norvartis animal health사의 전염성 조혈기괴사증의 예방을 위한 DNA 백신인 Apex-IHN, 일본에서 이리도바이러스의 불활화백신 등을 제외하면, 어류 바이러스로 인한 질병을 예방하기 위해 상용화된 백신은 거의 없으며 국내에서도 품목허가가 된 바이러스 백신은 일본에서 수입한 이리도 바이러스 불활화 백신 1종이다. 또한, 바이러스성 출혈성 패혈증의 백신은 출혈성 패혈증 바이러스의 구조와 항원단백질에 대한 연구, 이들 기초 연구를 바탕으로 불활화 백신, 단백질 재조합 백신, 약독화 백신, DNA 백신 등에 대한 연구가 수행되고 있으나, 아직까지 안정성 및 효능이 확보된 백신은 개발된 바 없다. 이는 어류 바이러스 백신의 제조를 위한 적절한 후보 바이러스의 탐색, 백신의 효과적인 제조 및 안정성 확보 등이 매우 어려움을 보여준다.Many vaccines have been developed to prevent disease caused by bacteria, but Pharmaq's vaccine against infectious pancreatic ulcer virus (IPNV) and DNA vaccine for the prevention of infectious hematopoietic necrosis from Norvartis animal health, Apex-IHN, Japan Except for inactivated vaccine of Irido virus, few vaccines are commercially available to prevent diseases caused by fish virus, and one of the Irido virus inactivated vaccines imported from Japan is a commercial vaccine. In addition, the vaccine of viral hemorrhagic sepsis has been studied on the structure and antigenic proteins of hemorrhagic sepsis virus, and based on these basic studies, studies on inactivating vaccines, recombinant protein vaccines, attenuated vaccines, DNA vaccines, etc. have been conducted. Until now, no vaccine has been developed that has secured stability and efficacy. This shows that it is very difficult to search for a suitable candidate virus for the production of a fish virus vaccine, to prepare the vaccine effectively and to ensure stability.
이에 본 발명자들은 국내에서 발생한 바이러스성 출혈성 패혈증을 분석하여 백신 후보주를 선발하고, 상기 후보주를 이용하여 넙치의 바이러스성 출혈성 패혈증을 예방하기 위한 최적의 백신을 개발함으로써 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by analyzing a viral hemorrhagic sepsis occurring in Korea, selecting a vaccine candidate, and developing an optimal vaccine for preventing viral hemorrhagic sepsis of the flounder using the candidate strain.
따라서 본 발명의 목적은 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신을 제공하는 것이다.It is therefore an object of the present invention to provide an inactivated vaccine for preventing viral hemorrhagic sepsis of olive flounder.
본 발명의 다른 목적은 상기 백신을 이용한 넙치의 바이러스성 출혈성 패혈증의 예방 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preventing viral hemorrhagic sepsis of olive flounder using the vaccine.
상기 목적을 달성하기 위하여, 본 발명은 불활성화된 바이러스성 출혈성 패혈증 바이러스 FP-VHS2010-01 항원(KCTC 12153BP)을 포함하는 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신을 제공한다.In order to achieve the above object, the present invention provides an inactivated vaccine for the prevention of viral hemorrhagic sepsis of the olive flounder containing inactivated viral hemorrhagic sepsis virus FP-VHS2010-01 antigen (KCTC 12153BP).
본 발명은 상기 백신을 넙치에 투여하는 것을 포함하는, 넙치의 바이러스성 출혈성 패혈증의 예방 방법을 제공한다.The present invention provides a method for preventing viral hemorrhagic sepsis of the flounder, comprising administering the vaccine to the flounder.
본 발명에 따른 백신은 넙치에서 발생하는 바이러스성 출혈성 패혈증을 효과적으로 예방할 수 있으므로, 넙치의 양식 산업 등에 유용하게 사용될 수 있다.Since the vaccine according to the present invention can effectively prevent viral hemorrhagic sepsis occurring in the flounder, it can be usefully used for the aquaculture industry of the flounder.
도 1 내지 4는 실시예 1에서 분리된 VHS 바이러스 FP-VHS2010-01의 당단백질 염기서열을 종래 알려진 VHS 바이러스인 NCBI AY167587 (NBCI), FP-VHS2005-01(2005년 제주 분리), FP-VHS2010-02(2010년 포항 분리) 및 FP-VHS2011-01(2011년 제주 분리)의 당단백질 염기서열과 비교한 것이다.1 to 4 shows glycoprotein sequences of the VHS virus FP-VHS2010-01 isolated in Example 1, NCBI AY167587 (NBCI), FP-VHS2005-01 (separated from Jeju, 2005), FP-VHS2010, which are known VHS viruses. This is compared with the glycoprotein sequences of -02 (2010 Pohang separation) and FP-VHS2011-01 (Jeju separation 2011).
도 5는 실시예 1에서 분리된 VHS 바이러스 FP-VHS2010-01을 다양한 농도로 다양한 크기의 넙치에 접종한 후 누적폐사율(%)을 나타낸 그래프이다.Figure 5 is a graph showing the cumulative mortality (%) after inoculation of the flounder of various sizes at various concentrations of the VHS virus FP-VHS2010-01 isolated in Example 1.
도 6은 실시예 1에서 분리된 VHS 바이러스 FP-VHS2010-01을 넙치에 접종한 후, 신장 조직 내 면역 유전자의 발현율을 확인한 결과이다. a는 조건 트리(condition tree)를, b는 산점도(scatter plot)를, c는 스캔 이미지를 나타낸다.Figure 6 shows the result of confirming the expression rate of immune genes in kidney tissue after inoculation on the flounder with the VHS virus FP-VHS2010-01 isolated in Example 1. a denotes a condition tree, b denotes a scatter plot, and c denotes a scanned image.
도 7은 실시예 1에서 분리된 VHS 바이러스 FP-VHS2010-01의 구조 단백질을 다클론 항체를 이용하여 (a) SDS-PAGE 및 (b) 웨스턴 블롯으로 분석한 사진이다. 각 도면에서 레인 M은 마커를, 레인 1은 VHS 바이러스 FP-VHS2010-01을 로딩한 것이다.Figure 7 is a photograph of the structural protein of VHS virus FP-VHS2010-01 isolated in Example 1 by (a) SDS-PAGE and (b) Western blot using a polyclonal antibody. In each figure, lane M is the marker and lane 1 is the VHS virus FP-VHS2010-01.
도 8은 실시예 1에서 분리된 VHS 바이러스 FP-VHS2010-01을 EPC 세포주에 접종한 후 다양한 온도에서 배양한 세포의 사진이다. a는 정상세포를, b 내지 d는 바이러스 접종 후 3일간 각각 15℃, 20℃, 및 25℃에서 배양한 세포를 나타낸다.8 is a photograph of cells cultured at various temperatures after inoculation of the VHS virus FP-VHS2010-01 isolated in Example 1 into an EPC cell line. a represents normal cells, and b to d represent cells cultured at 15 ° C., 20 ° C., and 25 ° C. for 3 days after virus inoculation, respectively.
도 9는 실시예 1에서 분리된 VHS 바이러스 FP-VHS2010-01을 EPC 세포주에 접종한 후, 각각 배양 플라스크(125cm2; a) 및 롤러 배양병(850cm2; b)에서 배양한 결과를 나타낸 것이다. 9 shows the results of incubating the VHS virus FP-VHS2010-01 isolated in Example 1 into the EPC cell line, followed by culturing in a culture flask (125 cm 2 ; a) and a roller culture bottle (850 cm 2 ; b), respectively. .
도 10은 각각 포르마린(a) 및 열(b)로 불활성화된 VHS 백신을 접종한 EPC 세포주를 2일간 배양한 후의 사진이다. 10 is a photograph after incubating EPC cell lines inoculated with VHS vaccine inactivated with formarin (a) and heat (b), respectively, for 2 days.
도 11은 백신 처리 방법 및 백신 처리시 넙치의 사육 수온에 따른 면역관련 유전자의 발현율 변화를 나타낸 사진이다. a는 고온 실험군을, b는 저온 실험군을, c는 대조군을 나타낸 것이며, a 및 b에서 레인 1 및 2는 백신침지군이고, 레인 3 및 4는 백신주사군이며, c에서 레인 1 및 2는 음성 대조군이고, 레인 3 및 4는 PBS 투여군이다.Figure 11 is a photograph showing a change in the expression rate of immune-related genes according to the breeding water temperature of the flounder during the vaccine treatment method and vaccine treatment. a is a high temperature experimental group, b is a low temperature experimental group, c is a control group, lanes 1 and 2 are vaccinated groups in a and b, lanes 3 and 4 are vaccine injection groups, and lanes 1 and 2 in c Negative control, lanes 3 and 4 are PBS administration group.
도 12는 백신 처리 방법 및 백신 처리시 넙치의 사육 수온에 따른 공격 실험 후의 넙치의 생존율을 나타낸 그래프이다.12 is a graph showing the survival rate of the flounder after the attack experiment according to the breeding water temperature of the flounder during the vaccine treatment method and vaccine treatment.
도 13은 본 발명의 백신 처리 후 공격실험을 실시할 경우의 평균 누적 폐사율(a), 라이소자임 활성(b), 및 VHSV에 대한 ELISA 결과를 나타낸 그래프이다.Figure 13 is a graph showing the mean cumulative mortality (a), lysozyme activity (b), and ELISA results for VHSV when the challenge test after the vaccine treatment of the present invention.
도 14는 본 발명의 백신 투여 후 넙치의 각 장기 조직을 H&E 염색으로 살펴본 사진이다.Figure 14 is a photograph of each organ tissue of the olive flounder after administration of the vaccine of the present invention by H & E staining.
본 발명은 불활성화된 바이러스성 출혈성 패혈증 바이러스 FP-VHS2010-01(KCTC 12153BP) 항원을 포함하는 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신을 제공한다. The present invention provides an inactivated vaccine for preventing viral hemorrhagic sepsis of olive flounder, which comprises the inactivated viral hemorrhagic sepsis virus FP-VHS2010-01 (KCTC 12153BP) antigen.
본 발명의 백신에 사용되는 바이러스성 출혈성 패혈증(VHS) 바이러스 FP-VHS2010-01은 거제의 양식장에서 넙치의 대량폐사를 일으킨 원인 바이러스로 분리된 균주로서, 종래 알려진 VHS 바이러스(GenBank에 등록된 AY 167587; 2005년 제주에서 분리된 FP-VHS2005-01; 2010년 포항에서 분리된 FP-VHS2010-02; 및 2011년 제주에서 분리된 FP-VHS2011-01)와 다른 균주임이 확인되었다(도 1 내지 4 참조). Viral hemorrhagic sepsis (VHS) virus used in the vaccine of the present invention FP-VHS2010-01 is a strain isolated from the causative virus that caused the mass mortality of the flounder in Geoje farm, a known VHS virus (AY 167587 registered in GenBank) ; FP-VHS2005-01 isolated from Jeju in 2005; FP-VHS2010-02 isolated from Pohang in 2010; and FP-VHS2011-01 isolated from Jeju in 2011) (see FIGS. 1 to 4). ).
상기 백신에 사용되는 균주인 VHS 바이러스 FP-VHS2010-01은 넙치에 2×107TCID50/마리, 2×106TCID50/마리 및 2×105TCID50/마리의 농도로 주사되는 경우, 주사 6일째 50% 이상의 폐사율을 나타내고, 주사 2주째에는 90-100%의 폐사율을 나타내며, 이러한 폐사율은 넙치의 크기가 작을수록, 그리고 바이러스의 농도가 높을수록 증가한다. 또한, 상기 VHS 바이러스 FP-VHS2010-01은 넙치에 감염시 감염되지 않은 넙치에 비해 면역 유전자를 3 내지 13배 이상 증가시킨다. 이는 상기 VHS 바이러스 FP-VHS2010-01이 병원성이 매우 높은 바이러스임을 보여준다. When the strain used for the vaccine, VHS virus FP-VHS2010-01, is injected into the flounder at the concentration of 2 × 10 7 TCID 50 /, 2 × 10 6 TCID 50 / and 2 × 10 5 TCID 50 / At 6 days of injection, mortality of 50% or more is shown, and mortality of 90-100% is shown at 2 weeks of injection, and this mortality increases with smaller flounder and higher virus concentration. In addition, the VHS virus FP-VHS2010-01 increases the immune gene by 3 to 13 times or more when compared with the flounder uninfected. This shows that the VHS virus FP-VHS2010-01 is a highly pathogenic virus.
본 발명에 따른 백신은 당업계에 통상적으로 알려진 제조 방법에 따라 제조될 수 있다. 상기 방법은 백신 균주의 배양, 바이러스 균주의 분리, 바이러스의 불활성화, 및 농축 및 희석 등의 과정을 포함한다. Vaccines according to the invention can be prepared according to manufacturing methods commonly known in the art. The method includes culturing the vaccine strain, isolation of the virus strain, inactivation of the virus, and concentration and dilution.
본 발명의 하나의 구체예에 있어서, 상기 VHSV 백신은 최적의 조건 중 하나로서 VHS 바이러스 FP-VHS2010-01을 18~22℃, 바람직하게는 20℃의 온도에서 배양함으로써 수득될 수 있다. 상기 온도 조건은 VHS 바이러스 FP-VHS2010-01의 최대 증식을 유도하여 본 발명의 백신 내에 항원을 증대시키는데 유리하다. 상기 바이러스의 배양을 위한 세포주로는 EPC 세포주가 사용될 수 있으나, 이에 제한되지 않으며 당업계에 알려진 통상적인 세포주, 예를 들어 BF-2, RTG-2, FHM, CHSE-214 등이 사용될 수 있다. In one embodiment of the invention, the VHSV vaccine can be obtained by culturing the VHS virus FP-VHS2010-01 at a temperature of 18 to 22 ℃, preferably 20 ℃ as one of the optimal conditions. Such temperature conditions are advantageous for inducing maximal proliferation of the VHS virus FP-VHS2010-01 to augment the antigen in the vaccine of the present invention. As the cell line for culturing the virus, an EPC cell line may be used, but is not limited thereto. Conventional cell lines known in the art, for example, BF-2, RTG-2, FHM, CHSE-214, and the like may be used.
다른 구체예에 있어서, 본 발명의 백신은 최적의 조건 중 하나로서 상기 배양된 VHS 바이러스를 열을 이용하여 불활성화시킴으로써, 바람직하게는 50-70℃의 온도하에서 2 내지 5일간 둠으로써, 수득될 수 있다. 본 발명의 백신은 포르마린을 이용하여 VHS 바이러스를 불활성화시켜 제조될 수도 있으나, 포르마린 불활성화는 사용된 포르마린을 제거하는 추가의 공정이 필요하며 상기 불활성화된 바이러스가 세포 재배양시 독성을 나타내므로, 열을 이용하여 불활성화시키는 것이 바람직하다(도 10 참조).In another embodiment, the vaccine of the present invention can be obtained by inactivating the cultured VHS virus with heat as one of the optimal conditions, preferably by leaving it for 2 to 5 days at a temperature of 50-70 ° C. Can be. The vaccine of the present invention may be prepared by inactivating the VHS virus using formarin, but formarin inactivation requires an additional process of removing used formarin and the inactivated virus is toxic in cell culture, It is preferable to deactivate using heat (see FIG. 10).
한편, 본 발명의 백신은 넙치의 크기가 13~17cm일 경우 주사에 의해 접종되는 것이 바람직하다. 백신 접종 방법으로 침지법이 알려져 있으나, 침지법의 경우 면역관련 유전자의 발현 증가 및 폐사율 면에서 주사법에 비해 낮은 효율을 나타낸다. 또한, 본 발명의 백신은 처리시 저온(예를 들어 10 내지 14℃)에 비해 고온(예를 들어 18 내지 22℃)에서 접종하는 것이 유리하다. 저온에서 처리하는 경우 면역관련 유전자의 발현 증가 및 폐사율 면에서 고온에서 처리하는 경우에 비해 낮은 효율을 나타낸다. 가장 바람직하게는, 본 발명의 백신은 고온/주사법에 의해 접종될 수 있다. On the other hand, the vaccine of the present invention is preferably inoculated by injection when the size of the flounder 13 ~ 17cm. Immersion is known as a vaccination method, but dipping shows lower efficiency than the injection method in terms of increased expression and mortality of immune-related genes. In addition, the vaccines of the present invention are advantageously inoculated at high temperatures (eg 18-22 ° C.) as compared to low temperatures (eg 10-14 ° C.) during treatment. The low temperature treatment shows lower efficiency than the high temperature treatment in terms of increased expression and mortality of immune-related genes. Most preferably, the vaccine of the present invention can be inoculated by high temperature / injection.
본 발명에 따른 불활성화 백신은 면역증강제(adjuvant)를 추가로 포함할 수 있다. 상기 면역증강제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있다. 또한, 안정화제 또는 첨가제로서, 당류나 아미노산류, 광물유, 식물유, 백반, 인산알루미늄, 벤토나이트, 실리카, 무라밀디펩티드(muramyl dipeptide) 유도체, 사이모신, 인터류킨 등이 사용될 수도 있다.Inactivated vaccines according to the invention may further comprise an adjuvant. The immunopotentiator may be used without limitation as long as it is known in the art. As the stabilizer or additive, sugars, amino acids, mineral oils, vegetable oils, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, thymosin, interleukin, and the like may also be used.
본 발명에 따른 백신은 적당한 용적의, 예를 들어 약 10~500 ㎖ 부피의 바이알에 백신액을 분주한 다음, 밀봉하여 사용될 수 있다. The vaccine according to the invention can be used by dispensing the vaccine solution in a vial of a suitable volume, for example about 10-500 ml volume, and then sealing.
나아가, 본 발명은 상기 백신을 넙치에 투여하는 것을 포함하는, 넙치의 바이러스성 출혈성 패혈증의 예방 방법을 제공한다. 상기 넙치는 감염의 위험성이 있는 넙치 등의 어류를 포함하다. Furthermore, the present invention provides a method for preventing viral hemorrhagic sepsis of the flounder, comprising administering the vaccine to the flounder. The flounder includes fish such as flounder which are at risk of infection.
본 발명의 불활성화 백신을 이용하여 넙치의 바이러스성 출혈성 패혈증을 예방하는 방법은 본 발명의 백신을 복강내 투여 등을 이용하여 넙치에 투여하는 단계를 포함한다. The method for preventing viral hemorrhagic sepsis of the olive flounder using the inactivated vaccine of the present invention includes administering the vaccine of the present invention to the flounder using intraperitoneal administration or the like.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
실시예 1: VHSV 백신 후보주 선발Example 1: VHSV Vaccine Candidate Selection
2010년 거제지역 넙치 양식장을 대상으로 PCR 방법 및 세포배양에 의해 바이러스성 출혈성 패혈증 바이러스(VHSV)를 검사하였다. 검사결과, 2개의 양식장에서 총 5마리의 넙치에서 5종의 VHSV가 발견되었다. 상기 5종의 VHSV에 대해 병원성 예비실험을 통해 가장 병원성이 높은 균주를 VHSV 백신을 위한 후보주로 선발하고, 이를 VHS 바이러스 FP-VHS2010-01으로 명명하였다. 상기 균주를 2012년 2월 19일에 한국생명공학연구원 생물자원센터에 KCTC 12153BP로 기탁하였다. Viral hemorrhagic sepsis virus (VHSV) was examined in PCR in 2010 and in flounder farms in Geoje. As a result, five VHSVs were found in five flounder in two farms. The most pathogenic strains were selected as candidate strains for the VHSV vaccine through the pathogenic preliminary experiments for the five VHSV, and named the VHS virus FP-VHS2010-01. The strain was deposited on February 19, 2012 as KCTC 12153BP at the Korea Institute of Bioscience and Biotechnology.
실시예 2: VHS 바이러스 FP-VHS2010-01의 당단백질 염기서열 분석Example 2: Glycoprotein Sequencing of VHS Virus FP-VHS2010-01
상기 실시예 1에서 선발한 VHS 바이러스 FP-VHS2010-01의 당단백질 염기서열을 분석하고, 이를 종래 알려진 VHS 바이러스(GenBank에 등록된 AY 167587; 2005년 제주에서 분리된 FP-VHS2005-01; 2010년 포항에서 분리된 FP-VHS2010-02; 및 2011년 제주에서 분리된 FP-VHS2011-01)와 비교하였다. The glycoprotein sequence of the VHS virus FP-VHS2010-01 selected in Example 1 was analyzed, and the known VHS virus (AY 167587 registered in GenBank; FP-VHS2005-01 isolated from Jeju in 2005; 2010 FP-VHS2010-02 isolated from Pohang and FP-VHS2011-01 isolated from Jeju in 2011).
구체적으로, 상기 VHS 바이러스 FP-VHS2010-01에 감염된 넙치의 신장 및 비장의 조직을 호모게나이저로 분쇄한 뒤 트리졸(trizol)을 이용하여 총 RNA(total RNA)를 분리하였다. 상기 분리된 총 RNA으로부터 cDNA를 합성한 후, 상기 cDNA를 주형(template)으로 하고 하기 서열번호 1 및 서열번호 2의 프라이머를 이용하여 RT-PCR을 수행하였다.Specifically, the kidney and spleen tissues of the flounder infected with the VHS virus FP-VHS2010-01 were crushed with a homogenizer and total RNA was isolated using trizol. After cDNA was synthesized from the isolated total RNA, the cDNA was used as a template, and RT-PCR was performed using primers of SEQ ID NO: 1 and SEQ ID NO: 2.
- VHSG-exp-F: 5'-GGATCCATGGAATGGAATACTTTTTCCTTGGTG-3' (서열번호 1);VHSG-exp-F: 5'-GGATCCATGGAATGGAATACTTTTTCCTTGGTG-3 '(SEQ ID NO: 1);
- VHSG-exp-R: 5'-AAGCTTGACCGTCTGACTTCTGGAGAA-3' (서열번호 2)VHSG-exp-R: 5'-AAGCTTGACCGTCTGACTTCTGGAGAA-3 '(SEQ ID NO: 2)
RT-PCR은 1X PCR 완충액(20 mM Tris-hydrochloride(pH 8.4), 50 mM KCl 및 1.5 mM MgCl2), 200 uM dNTP(Bioneer, Korea), 0.2 uM 프라미머 및 2U Taq DNA 중합효소(Bioneer, Korea) 및 50 ng의 주형 cDNA를 혼합한 후, 94℃, 5분 반응 후 94℃에서 1분, 55℃에서 1분 및 72℃에서 1분간 30회 반응시킨 다음 72℃에서 7분간 반응시켜 수행하였다. 상기 증폭된 PCR 생성물을 1X TAE(40 mM Tris-acetate, 1 mM EDTA) 완충액 상에서 1.5% 아가로스 겔에 로딩한 후, 100 V에서 15분간 전기영동하여 크기를 확인하였다. 상기 PCR 산물을 겔로부터 분리한 후 QIAquick 겔 추출 키트(Qiagen, USA)를 이용하여 정제하였다. 상기 정제된 DNA를 pGEM-T easy 벡터(Promega, pGEM-T easy vector system, USA)에 삽입한 후, 상기 플라스미드를 JM109(Takara, Japan) 컴피턴트 세포에 열충격에 의해 형질전환시켰다. 상기 형질전환된 세포를 2%(w/v) X-gal 40 ㎕, 20%(w/v) IPTG 용액 7 ㎕, 암피실린 50 ㎍/㎖이 포함된 LB 배지에 도말하여 37℃에서 18시간 배양한 후 PCR 산물 유전자가 삽입된 백색 콜로니를 선택하였다. 상기 콜로니를 50 ㎍/㎖ 암피실린(Sigma, USA)이 첨가된 LB 배지(Yeasy extract 5 g/ℓ, pancreatic digest of casein 10 g/ℓ, sodium chloride 10 g/ℓ, Difco;Miller)에서 대량 배양한 후 원심분리한 후, 플라스미드를 얻어 제한효소(EcoRI)로 절단하여 삽입된 DNA의 유무와 크기를 확인하였다. 정확한 크기를 갖는 DNA의 염기서열을 분석한 후, 2005년 제주에서 분리된 VHS 바이러스 및 2011년 제주에서 분리된 VHS 바이러스의 당단백질 염기서열과 비교하였다. 상기 비교 결과를 도 1 내지 4에 나타내었다. 비교 결과, VHS 바이러스 FP-VHS2010-01의 당단백질은 종래 알려진 VHS 바이러스(GenBank에 등록된 AY 167587; 2005년 제주에서 분리된 FP-VHS2005-01; 2010년 포항에서 분리된 FP-VHS2010-02; 및 2011년 제주에서 분리된 FP-VHS2011-01)와 98 내지 99%의 상동성을 나타내는 새로운 균주임이 확인되었다. RT-PCR contains 1 × PCR buffer (20 mM Tris-hydrochloride (pH 8.4), 50 mM KCl and 1.5 mM MgCl 2 ), 200 uM dNTP (Bioneer, Korea), 0.2 uM primer and 2U Taq DNA polymerase (Bioneer, Korea) and 50 ng of the template cDNA was mixed, followed by reaction at 94 ° C. for 5 minutes, 1 minute at 94 ° C., 1 minute at 55 ° C., and 1 minute at 72 ° C. for 30 minutes, followed by 7 minutes at 72 ° C. It was. The amplified PCR product was loaded on 1.5% agarose gel in 1X TAE (40 mM Tris-acetate, 1 mM EDTA) buffer, and then electrophoresed at 100 V for 15 minutes to confirm the size. The PCR product was separated from the gel and purified using QIAquick gel extraction kit (Qiagen, USA). The purified DNA was inserted into a pGEM-T easy vector (Promega, pGEM-T easy vector system, USA), and the plasmid was transformed by thermal shock to JM109 (Takara, Japan) competent cells. The transformed cells were plated in LB medium containing 40 μl of 2% (w / v) X-gal, 7 μl of 20% (w / v) IPTG solution, and 50 μg / ml of ampicillin and incubated at 37 ° C. for 18 hours. Then, white colonies into which the PCR product gene was inserted were selected. The colonies were incubated in LB medium (Yeasy extract 5 g / L, pancreatic digest of casein 10 g / L, sodium chloride 10 g / L, Difco; Miller) to which 50 μg / ml ampicillin (Sigma, USA) was added. After centrifugation, plasmids were obtained and digested with restriction enzymes (EcoRI) to confirm the presence and size of the inserted DNA. After analyzing the base sequence of the DNA having the correct size, it was compared with the glycoprotein base sequence of the VHS virus isolated from Jeju in 2005 and the VHS virus isolated from Jeju in 2011. The comparison results are shown in FIGS. 1 to 4. As a result, the glycoprotein of the VHS virus FP-VHS2010-01 was selected from the known VHS virus (AY 167587 registered with GenBank; FP-VHS2005-01 isolated from Jeju in 2005; FP-VHS2010-02 isolated from Pohang in 2010; And FP-VHS2011-01) isolated from Jeju in 2011 was confirmed to be a new strain showing a homology of 98 to 99%.
실시예 3: VHS 바이러스 FP-VHS2010-01의 병원성 분석Example 3: Pathogenicity Analysis of VHS Virus FP-VHS2010-01
상기 실시예 1에서 선발한 VHS 바이러스 FP-VHS2010-01의 병원성을 살펴보기 위하여, 20℃에 적응시킨 EPC 세포주(ATCC No; CRL-2872)를 25cm2 배양 플라스크에 계대배양한 다음 12시간 후 TCID 107/ml의 바이러스 용액 200μL를 접종(배지 5 mL)하여 배양하였다. 이후 하기와 같이 특성을 분석하였다.In order to examine the pathogenicity of the VHS virus FP-VHS2010-01 selected in Example 1, an EPC cell line (ATCC No; CRL-2872) adapted to 20 ° C. was passaged in a 25 cm 2 culture flask, followed by TCID 12 hours later. Incubated by inoculating 200 μL of 10 7 / ml virus solution (medium 5 mL). Then, the characteristics were analyzed as follows.
<3-1> 바이러스 농도에 따른 병원성 실험<3-1> Pathogenicity test according to virus concentration
상기 배양한 바이러스 FP-VHS2010-01을 원심분리한 후 108 TCID50/ml로 희석하여 2×107TCID50/마리, 2×106TCID50/마리의 농도가 되도록 각각 10마리의 넙치에 복강주사법으로 인위 감염 후, 10-12℃에서 2주간 누적폐사율을 조사하였다. 상기 실험 결과, 전 실험군에서 주사 6일째 넙치의 50% 이상이 폐사하였고, 주사 2주째에는 넙치의 90-100%가 폐사하였다. The cultured virus FP-VHS2010-01 was centrifuged and diluted to 10 8 TCID 50 / ml on 10 flounder so that the concentrations were 2 × 10 7 TCID 50 / head and 2 × 10 6 TCID 50 / head. The cumulative mortality was investigated for 2 weeks at 10-12 ℃ after artificial infection by intraperitoneal injection. As a result of the experiment, more than 50% of the flounder died on the 6th day of injection, and 90-100% of the flounder died on the 2nd week of injection.
<3-2> 바이러스 농도 및 넙치 크기에 따른 병원성 실험<3-2> Pathogenicity test according to virus concentration and flounder size
20g, 45g, 100g 및 200g의 특별한 이상 증상을 나타내지 않는 건강한 넙치를 각각 10마리씩 준비한 다음, 상기 실시예 <3-1>에서와 동일한 방법으로 VHS 바이러스 FP-VHS2010-1을 복강내로 인위 감염시킨 후 10-12℃에서 17일간 누적폐사율을 조사하였다. 20g 실험군에는 2×105TCID50/마리 및 1×104TCID50/마리의 농도로 주사하였고, 45g 실험군에는 4×105TCID50/마리 및 2.5×104TCID50/마리의 농도로 주사하였고, 100g 실험군에는 1×106TCID50/마리의 농도로 주사하였으며, 200g 실험군에는 2×106TCID50/마리의 농도로 주사하였다.After preparing 10 healthy flounder each showing 20 g, 45 g, 100 g and 200 g without any unusual abnormalities, and then artificially infected the VHS virus FP-VHS2010-1 in the same manner as in Example <3-1>. Cumulative mortality was examined at 10-12 ° C for 17 days. 20g group is 2 × 10 5 TCID 50 / mouse, and 1 × 10 4 TCID was injected at a concentration of 50 / mouse, 45g group is 4 × 10 5 TCID 50 / animal and 2.5 × 10 4 TCID injection at a concentration of 50 / mouse The 100g test group was injected at a concentration of 1 × 10 6 TCID 50 / head, and the 200g test group was injected at a concentration of 2 × 10 6 TCID 50 / head.
실험 결과, 20g 실험군(2×10TCID50
5/마리)은 1주에 50% 폐사율 및 2주에 80% 폐사율을 나타내었고, 45g 실험구(4×105TCID50/마리)은 1주에 20% 폐사율 및 2주에 60% 폐사율을 나타내었으며, 100g 실험군(1×106TCID50/마리)은 1주에 0% 폐사율 및 2주에 30% 폐사율을 나타내었고, 200g 실험군(2×106TCID50/마리)은 1주에 0% 폐사율 및 2주에 14% 폐사율을 나타내었다(도 5 참조). 상기 결과는 어체의 크기가 작을수록 바이러스의 병원성이 높음을 보여준다.As a result, the 20g experimental group (2 × 10 TCID 50 5 / mice) showed 50% mortality at 1 week and 80% mortality at 2 weeks, and the 45g experimental group (4 × 10 5 TCID 50 / head) was 20 at 1 week. % Mortality and 60% mortality at 2 weeks, 100g experimental group (1 × 10 6 TCID 50 per animal) showed 0% mortality at 1 week and 30% mortality at 2 weeks, and 200g experimental group (2 × 10 6) TCID 50 per animal) showed 0% mortality at 1 week and 14% mortality at 2 weeks (see FIG. 5). The results show that the smaller the size of the fish, the higher the pathogenicity of the virus.
한편, 바이러스의 농도를 달리하여 실험한, 20g 실험군(1×104TCID50/마리)은 1주에 20% 폐사율 및 2주에 50% 폐사율을 나타내었고, 45g 실험군(2.5×104TCID50/마리)은 1주에 0% 및 2주에 20%의 폐사율을 나타내어, 바이러스의 농도가 높을수록 병원성이 높음을 알 수 있었다(도 5 참조).On the other hand, the 20g experimental group (1 × 10 4 TCID 50 / head), tested at different concentrations of virus, showed 20% mortality at 1 week and 50% mortality at 2 weeks, and 45g experimental group (2.5 × 10 4 TCID 50). / Mari) showed mortality of 0% at 1 week and 20% at 2 weeks, indicating that the higher the concentration of virus, the higher the pathogenicity (see FIG. 5).
<3-3> VHS 바이러스 감염에 따른 넙치의 면역 유전자 발현율 변화 확인<3-3> Change of Immune Gene Expression Rate of Olive Flounder by VHS Virus Infection
실시예 1의 VHS 바이러스의 감염으로 인한 넙치의 면역 유전자 발현율 변화를 살펴보기 위하여, DNA 칩 마이크로어레이(자체 제작)을 이용하여 상기 바이러스가 감염된 넙치의 신장 조직에서의 면역 유전자의 발현 양상을 시간별로 확인하였다. 구체적으로, 12℃에서 10 cm의 넙치의 복강에 VHS 바이러스 FP-VHS2010-01을 접종한 후 1일 및 3일째 신장과 비장의 면역 유전자의 발현량을 대조군(정상 소견의 넙치의 신장과 비장의 면역 유전자의 발현량)과 비교하였다.In order to examine the changes in the immune gene expression rate of the flounder due to the infection of the VHS virus of Example 1, the expression patterns of the immune genes in the kidney tissues of the flounder infected with the virus were analyzed using DNA chip microarrays (self-made). Confirmed. Specifically, after inoculation of the VHS virus FP-VHS2010-01 into the abdominal cavity of a 10 cm flounder at 12 ° C., the expression levels of the immune genes of the kidney and the spleen were measured in the control group (the kidney and spleen of the flounder of normal findings). Expression level of immune genes).
도 6의 결과로부터 바이러스 감염 1일과 3일 후에 CD83, VHSV 유도 단백질 6, CD9, 카텝신(cathepsin) L 프리프로단백질(preproprotein), 인터페론 유도성 단백질 56, 리소자임 C 전구체, IFI56 등의 발현량이 대조구에 비해 3배 내지 13배 이상 증가하는 발현 양상을 확인할 수 있었다. 상기 결과는 VHS 바이러스 FP-VHS2010-1이 여러 면역 유전자의 발현을 증가시키는 고병원성 바이러스임을 보여준다.From the results of FIG. 6, the expression levels of CD83, VHSV-induced protein 6, CD9, cathepsin L preproprotein, interferon-induced protein 56, lysozyme C precursor, IFI56, and the like after 1 and 3 days of virus infection It was confirmed that the expression pattern increased by 3 to 13 times more than. The results show that the VHS virus FP-VHS2010-1 is a highly pathogenic virus that increases the expression of several immune genes.
실시예 4: 바이러스 백신 후보주의 구조 단백질 분석Example 4 Structural Protein Analysis of a Vaccine Vaccine Candidate
상기 실시예 1에서 선발된 바이러스 균주 FP-VHS2010-01의 구조 단백질을 분석하기 위하여, 하기와 같이 SDS-PAGE 및 웨스턴 블롯을 수행하였다. In order to analyze the structural protein of the virus strain FP-VHS2010-01 selected in Example 1, SDS-PAGE and Western blot was performed as follows.
<4-1> 바이러스 분리 및 정제<4-1> Virus Isolation and Purification
바이러스를 분리 및 정제하기 위하여, 대량 배양된 바이러스액의 상징액에 7% 폴리에틸렌 글리콜(PGE-6000) 및 2.3%의 NaCl을 첨가한 다음 4℃에서 교반하면서 하룻밤동안 배양하였다. 상기 배양물을 20,000g에서 40분 동안 원심분리하여 펠렛을 얻고, 이를 소량의 TNE 완충액(0.01M Tris HCl, 0.1M NaCl, 0.001M RDTA)에 재현탁시킨 다음 15% 수크로오스 쿠션(sucrose cushion) 위에 중축하고 4℃, 115,000g에서 1시간 동안 초고속 원심 분리하여 바이러스 펠렛을 얻었다. 이를 TNE 완충용액에 재현탁하여 20, 35 및 50% 수크로오스 불연속 구배 상에서 80,000g로 1.5시간 동안 원심분리하여 20%와 35% 경계지점에 형성된 바이러스 밴드를 회수하였다. 이를 TNE 완충용액에 재현탁한 다음, 115,000g에서 1시간 동안 원심분리하여 바이러스를 정제하였다.To isolate and purify the virus, 7% polyethylene glycol (PGE-6000) and 2.3% NaCl were added to the supernatant of the mass cultured virus solution, and then incubated overnight with stirring at 4 ° C. Centrifuge the culture at 20,000 g for 40 minutes to obtain pellets, resuspend in small amount of TNE buffer (0.01 M Tris HCl, 0.1 M NaCl, 0.001 M RDTA) and then on a 15% sucrose cushion Virus pellets were condensed and centrifuged at 115,000 g at 4 ° C. for 1 hour to obtain virus pellets. It was resuspended in TNE buffer and centrifuged at 80,000 g for 1.5 hours on 20, 35 and 50% sucrose discontinuous gradient to recover virus bands formed at the 20% and 35% boundaries. The virus was resuspended in TNE buffer and then purified by centrifugation at 115,000 g for 1 hour.
<4-2> SDS-PAGE<4-2> SDS-PAGE
FP-VHS2010-01 균주의 항원 단백질체 분석을 위하여 SDS-PAGE 분석을 수행하였다. 분리된 바이러스를 10분간 초음파로 분쇄한 다음, 2× 샘플 버퍼(0.12M Tris-HCl, 4% SDS, 20% glycerol, 5 mM 2-mercaptoethanol)와 1:1로 혼합하고 100℃에서 10분간 끊여서 준비하였다. 12% SDS-폴리아크릴아미드 겔에 준비된 시료를 15 ㎕ 로딩하고 100 V에서 1시간 20분 동안 전기영동하였다. 전기영동 끝난 겔을 염료(coomassie brilliant blue R-250)로 염색하고 탈색하여 관찰하였다.SDS-PAGE analysis was performed for analysis of the antigenic protein of the FP-VHS2010-01 strain. The virus was pulverized by sonication for 10 minutes, then mixed 1: 1 with 2 × sample buffer (0.12M Tris-HCl, 4% SDS, 20% glycerol, 5 mM 2-mercaptoethanol) and cut off at 100 ° C. for 10 minutes. Ready. 15 μl of the sample prepared on a 12% SDS-polyacrylamide gel was loaded and electrophoresed at 100 V for 1 hour 20 minutes. The gel was electrophoresed and stained with dye (coomassie brilliant blue R-250) and bleached to observe.
<4-3> 토끼 항체 제작<4-3> Rabbit antibody preparation
2주간 안정화한 토끼 2마리에 0주째에 본 발명의 바이러스액을 주사하였다. 4주째, 6주째 및 8주째에 동일한 바이러스액으로 1차 내지 3차 부스트(boost) 주사를 수행하였다. 이후 토끼 혈청 10 mL으로부터 단백질 A 컬럼을 이용하여 IgG를 정제하였다. 이후 항원이 결합된 친화성 컬럼을 이용하여 항원 특이적 항체를 정제하였다. 상기 정제된 항체를 투석 및 농축시켰다. 상기 정제된 항체에 FITC를 결합시키기 위하여, 항체 2 mg을 투석한 다음, FITC와 혼합하여 2시간 동안 교반하였다. 결합되지 않은 자유 FITC를 제거하기 위해 투석을 수행함으로써 FITC 결합된 항체를 수득하였다.Two rabbits stabilized for two weeks were injected with the virus solution of the present invention at week zero. At 4 weeks, 6 weeks and 8 weeks, the first to third boost injections were performed with the same virus solution. IgG was then purified using a Protein A column from 10 mL of rabbit serum. The antigen specific antibody was then purified using an affinity column to which the antigen was bound. The purified antibody was dialyzed and concentrated. To bind FITC to the purified antibody, 2 mg of antibody was dialyzed, then mixed with FITC and stirred for 2 hours. Dialysis was performed to remove unbound free FITC to obtain an FITC bound antibody.
<4-4> 웨스턴 블롯팅<4-4> Western blotting
실시예 <4-2>에서 SDS 전기영동한 겔에 니트로셀룰로오소 멤브레인 페이퍼(nitrocelluolse menbrane paper, NC paper, Amersham Biosciences, Germany)를 얹어서 100 V에서 1시간 동안 이전시키고, 상기 NC 페이퍼를 트윈(Tween 20)이 포함된 PBS(T-PBS)로 5회 세척한 다음 1% BSA(Bovine Serum Albumin, Sigma)로 1시간 동안 블랏킹(blocking)하고 T-PBS로 5회 세척하였다. 상기 실시예 <4-3>에서 제작한 VHSV 항체를 1/10,00로 희석하여 2시간 반응 후 T-PBS로 5회 세척하였다. 2차 항체로는 알칼라인 포스파테이트와 결합된 염소 항-토끼 면역글로불린(Sigma)을 사용하였으며, 이를 1/5,000로 희석하여 1 시간 동안 반응시킨 후 T-PBS로 5회 세척하고, BICP/NBT 포스파타아제 기질(Sigma)을 첨가하여 발색시켜 관찰하였다.In Example <4-2>, the SDS electrophoresis gel was placed on a nitrocellulose membrane paper (nitrocelluolse menbrane paper, NC paper, Amersham Biosciences, Germany) and transferred at 100 V for 1 hour, and the NC paper was twined ( Tween 20) was washed five times with PBS (T-PBS) containing, and then blocked with 1% BSA (Bovine Serum Albumin, Sigma) for 1 hour and washed five times with T-PBS. The VHSV antibody prepared in Example <4-3> was diluted to 1 / 10,00 and washed 5 times with T-PBS after 2 hours of reaction. As a secondary antibody, goat anti-rabbit immunoglobulin (Sigma) coupled with alkaline phosphate was used, diluted to 1 / 5,000, reacted for 1 hour, washed five times with T-PBS, and BICP / NBT. Observation was carried out by the addition of phosphatase substrate (Sigma).
상기 SDS-PAGE 결과를 도 7의 (a)에 나타내었고, 웨스턴 블롯 결과를 도 7의 (b)에 나타내었다. 각 도의 M 레인은 마커를 의미하고, 1 레인은 FP-VHS2010-01을 로딩한 결과이다. 도 7(a)에 나타난 본 발명의 VHS 바이러스 FP-VHS2010-01의 구조 단백질을 종래 보고된 문헌에 기재된 VHS 바이러스(김수미, 양식넙치, Paralichthys olivaceus에서 발생하는 viral hemorrhagic septicemia virus (VHSV)감염증에 관한 연구, 부경대학교 대학원 어병학과, 이학박사 학위논문, p.101)와 비교한 결과, 서로 같은 단백질 패턴을 나타내었고 다른 바이러스 HIRRV 등과는 다른 단백질 패턴을 나타내었다. 또한, 도 7(b)의 결과로부터 FP-VHS2010-01 바이러스가 항원-항체 반응에서 항원성이 있음을 확인할 수 있었다. The SDS-PAGE results are shown in (a) of FIG. 7, and the Western blot results are shown in (b) of FIG. 7. Lane M in each figure means a marker, and lane 1 is the result of loading FP-VHS2010-01. The structural protein of the VHS virus FP-VHS2010-01 of the present invention shown in FIG. 7 (a) relates to a VHS virus (Kim Soo-mi, cultured flounder, viral hemorrhagic septicemia virus (VHSV) infection occurring in Paralichthys olivaceus). Study, the Department of Fish Pathology, Graduate School, Pukyong National University, Ph.D., p.101) showed the same protein pattern and different protein patterns from other viral HIRRVs. In addition, the results of Figure 7 (b) it was confirmed that the FP-VHS2010-01 virus antigenic in the antigen-antibody reaction.
실시예 5: 백신 제조방법에 따른 효율 분석Example 5 Efficiency Analysis According to Vaccine Preparation Method
실시예 1에서 선발된 VHS 바이러스 FP-VHS2010-01에 대한 최적의 백신을 제조하기 위해, 하기와 같이 백신 제조방법에 따른 효율을 분석하였다. In order to prepare the optimal vaccine against the VHS virus FP-VHS2010-01 selected in Example 1, the efficiency according to the vaccine preparation method was analyzed as follows.
<5-1> 배양 온도에 따른 VHS 바이러스의 증식 분석<5-1> Proliferation Analysis of VHS Virus According to Culture Temperature
VHS 바이러스 FP-VHS2010-01을, 해산어 유래 VHSV의 증식율이 가장 좋다고 알려진 EPC 세포주에 접종하고 다양한 온도 조건(15℃, 20℃ 및 25℃) 하에서 3일간 배양한 후, 세포의 증식을 분석하였다. 구체적으로, EPC 세포주를 1% 항생제(Gibco) 및 10% FBS(Gibco)가 첨가된 MEM 배지(welgene)에서 단층 배양한 다음, 상기 배양된 EPC 세포주에 VHS 바이러스를 접종하고 실온에서 30분간 흡착시켰다. 이후 5% FBS와 1% 항생제를 첨가한 MEM 배지를 첨가한 다음, 15℃, 20℃ 및 25℃의 온도에서 배양하면서 세포변성 효과(cytopathic effect, CPE)를 역상현미경을 이용하여 관찰하였다. 대조군으로 PBS를 접종한 정상세포를 사용하였다. VHS virus FP-VHS2010-01 was inoculated into EPC cell lines known to have the highest proliferation rate of marine fish-derived VHSV and incubated under various temperature conditions (15 ° C, 20 ° C and 25 ° C) for 3 days, and then cell proliferation was analyzed. . Specifically, EPC cell lines were cultured monolayer in MEM medium (welgene) to which 1% antibiotic (Gibco) and 10% FBS (Gibco) were added, and then the cultured EPC cell lines were inoculated with VHS virus and adsorbed at room temperature for 30 minutes. . After adding MEM medium with 5% FBS and 1% antibiotics, the cytopathic effect (CPE) was observed using an inverted microscope while culturing at a temperature of 15 ℃, 20 ℃ and 25 ℃. Normal cells inoculated with PBS were used as a control.
상기 실험결과를 도 8에 나타내었다. 도 8의 a는 대조군을, b 내지 d는 바이러스 접종 후 각각 15℃, 20℃ 및 25℃에서 배양한 세포주의 사진이다. 상기 도에서 보는 바와 같이, EPC 세포주는 25℃에서 가장 성장이 빠른데 반해 바이러스의 증식은 20℃에서 가장 높은 세포변성 효과를 나타내었다. 이는 통상적인 세포주 배양 온도와 바이러스 증식 온도가 다르다는 것을 보여준다. The experimental results are shown in FIG. 8. 8 a is a control, b to d are photographs of cell lines cultured at 15 ° C., 20 ° C. and 25 ° C. after virus inoculation, respectively. As shown in the figure, EPC cell lines showed the fastest growth at 25 ° C, whereas virus proliferation showed the highest cytopathic effect at 20 ° C. This shows that the virus cell growth temperature is different from the normal cell line culture temperature.
<5-2> 배양 용기 크기에 따른 증식 분석<5-2> Proliferation analysis according to the culture vessel size
배양 용기 크기에 따른 증식의 차이를 분석하기 위하여, 바이러스가 포함된 300 mL의 배양액을 배양 플라스크(125cm2; 도 9a) 및 롤러 배양병(roller bottle, 850cm2; 도 9b)에서 각각 배양하였다. In order to analyze the difference in proliferation according to the culture vessel size, 300 mL of the culture solution containing the virus was incubated in a culture flask (125 cm 2 ; FIG. 9A) and a roller bottle (850 cm 2 ; FIG. 9B), respectively.
동일한 양(300 mL)을 배양하더라고 면적이 큰 롤러 배양병에서 배양하는 것이 더 높은 농도의 바이러스 배양액을 제조하는데 유리한 것으로 확인되었다. Even incubating the same amount (300 mL), culturing in a large roller culture bottle was found to be advantageous for producing higher concentration of virus culture.
<5-3> 바이러스 불활성화 방식에 따른 백신의 안정성 검사<5-3> Stability test of vaccine according to virus inactivation method
백신의 제조를 위한 최적의 불활성화 방식을 확인하기 위하여, 실시예 1에서 선발한 VHS 바이러스를 통상적으로 바이러스 불활성화에 사용되는 방법인 포르마린 불활성화 방법과 열 불활성화 방법을 통해 불활성화한 후, 세포를 재배양하여 안정성을 비교하였다. 구체적으로, 포르마린 불활성화 방법은 냉동된 바이러스 배양액을 해동시켜 원심분리(40,000 rpm, 4℃, 10분)하여 세포 성분을 제거하고, 상층액에 1:4,000(0.4%)의 비율로 포르말린을 처리하여 37℃에서 3일간 반응시켜 수행하였다. 열 불활성화 방법은 세포성분을 제거한 상기 상층액을 60℃에서 3일간 불활성화시켜 수행하였다. 상기 제조된 백신은 4℃에 보관한 상태로 사용하였다. In order to confirm the optimal inactivation method for the preparation of the vaccine, the VHS virus selected in Example 1 was inactivated through a formalin inactivation method and a thermal inactivation method, which is a method commonly used for virus inactivation. Cells were cultured to compare stability. Specifically, the formalin inactivation method is thawed frozen virus cultures by centrifugation (40,000 rpm, 4 ℃, 10 minutes) to remove the cell components, the supernatant treated with formalin at a ratio of 1: 4,000 (0.4%). The reaction was carried out at 37 ° C. for 3 days. The heat inactivation method was performed by inactivating the supernatant from which cell components were removed at 60 ° C. for 3 days. The prepared vaccine was used while stored at 4 ℃.
상기 실험결과를 도 10에 나타내었다. 도 10의 a는 본 발명의 바이러스를 포르마린으로 불활성화한 후 EPC 세포에 접종하여 2일간 배양한 세포의 사진이며, 도 10의 b는 본 발명의 바이러스를 열로 불활성화한 후 EPC 세포에 접종하여 2일간 배양한 세포의 사진이다. 상기 도에서 보는 바와 같이, 포르마린으로 불활성화된 바이러스는 포르말린 독성으로 인한 세포변성을 나타내어 안전성에 있어 문제가 있는 것으로 확인되었고, 또한 포르마린을 제거하는 추가의 과정(불활화된 반응물을 원심분리(X50,000g, 초원심분리, 4℃, 1시간)하여 얻은 펠렛을 FBS가 없는 MEM에 재부유시킨 다음 보존하는 과정)을 추가한다면, 이로 인해 바이러스의 손실 및 경제적 손실이 생기며, 나아가 불활화된 바이러스가 세포 재배양시 독성을 나타내는 문제점이 있다. 이에 반해, 열로 불활성화된 바이러스는 세포 변성이 없어 안전하므로, 제조 후 바로 사용할 수 있으므로 열 불활성화 방법이 본 발명의 백신 제조에 효과적인 것으로 판단되었다. The experimental results are shown in FIG. 10. 10 a is a photograph of cells cultured for 2 days by inoculating EPC cells after inactivating the virus of the present invention with formarin, and FIG. 10 b is inoculating EPC cells after inactivating the virus of the present invention with heat. It is a photograph of the cells cultured for 2 days. As shown in the figure, the virus inactivated by formalin was found to be cytotoxic due to formalin toxicity, which was confirmed to be a problem in safety, and also a further process of removing formalin (inactivated reactant by centrifugation (X50). , 000 g, ultracentrifugation, 4 ° C., 1 hour), resuspending pellets in MEM without FBS and then preserving them), resulting in virus loss and economic loss. There is a problem of toxicity in cell culture. In contrast, heat-inactivated viruses are safe from cell degeneration and can be used immediately after preparation. Therefore, it has been determined that the heat inactivation method is effective for the vaccine preparation of the present invention.
실시예 6: 백신의 처리방법에 따른 효율 분석Example 6 Analysis of Efficiency According to Vaccine Treatment
상기 실시예 5에서 열 불활성화 방법에 의해 제조된 백신을 대상으로, 백신의 처리방법 및 처리 온도가 면역능에 미치는 영향을 하기와 같이 분석하였다. 넙치(평균체중 24.6g, 13.4cm)를 20℃에서 사육한 고온 실험군과 12℃에 사육한 저온 실험군으로 나눈 다음, 주사백신(106 TCID50/마리, 복강주사)과 침지백신(106 TCID50/마리, 3분간 침지, 1회 실시)을 처리하였으며, 12℃에서 사육하면서 2주 후 각 실험군별로 20마리씩 VHS 바이러스를 접종하였다. 상기 접종 후 면역관련 유전자의 발현 및 누적폐사율을 조사하였다. For the vaccine prepared by the heat inactivation method in Example 5, the effect of the treatment method and treatment temperature of the vaccine on the immune capacity was analyzed as follows. The flounder (average weight 24.6g, 13.4cm) was divided into high-temperature experimental group bred at 20 ° C and low-temperature experimental group bred at 12 ° C, followed by injection vaccine (10 6 TCID 50 / head, intraperitoneal injection) and immersion vaccine (10 6 TCID 50 / horses, soaked for 3 minutes, carried out once), and was inoculated with VHS virus 20 animals for each experimental group 2 weeks after breeding at 12 ℃. Expression and cumulative mortality of immune-related genes were investigated after the inoculation.
면역관련 유전자(GAPDH, Mx, IFN 및 ISG-15)의 발현량을 하기와 같이 측정하였다. 백신을 투여한지 2주 후의 넙치를 무작위로 선정하여 RNA를 추출하였다. 상기 추출한 RNA를 대상으로, 하기 표 1에 기재된 각 면역관련 유전자용 프라이머 및 조건을 이용하여 RT-PCR을 수행하였다. Expression levels of immune related genes (GAPDH, Mx, IFN and ISG-15) were measured as follows. Two weeks after the administration of the vaccine, the flounder was randomly selected and RNA was extracted. The extracted RNA was subjected to RT-PCR using primers and conditions for each immune-related gene described in Table 1 below.
표 1
Table 1
유전자명 | 프라이머 이름 | 염기서열 | 조건 | |
GAPDH | GAPDH-1U | 5'-ccaggtcgtctccacagact-3'(서열번호: 3) | 94℃, 5분94℃, 20초 27 사이클55℃, 30초72℃, 30초72℃, 7분 | |
GAPDH-1D | 5'-catgagaccagcttgacgaa-3'(서열번호: 4) | |||
IFN | IFN-1U | 5'-tacagccaggtgtcaaatgc-3'(서열번호: 5) | 94℃, 5분94℃, 20초 29 사이클59℃, 30초72℃, 30초72℃, 7분 | |
IFN-1D | 5'-tggaatcctcctcaaacagg-3'(서열번호: 6) | |||
Mx | floMx-F | 5'-aacagccaaggcaaagattg-3'(서열번호: 7) | 94℃, 5분94℃, 20초 29 사이클59℃, 30초72℃, 30초72℃, 7분 | |
floMx-R | 5'-aatgtccagctcctccttca-3'(서열번호: 8) | |||
ISG-15 | ISG-15-F | 5'-ctccatgtaatctgcagcaa-3'(서열번호: 9) | 94℃, 5분94℃, 20초 26 사이클57℃, 30초72℃, 30초72℃, 7분 | |
ISG-15-R | 5'-cagatctagtgcaggtgtga-3'(서열번호: 10) |
Gene name | Primer name | Sequence | Condition | |
GAPDH | GAPDH-1U | 5'-ccaggtcgtctccacagact-3 '(SEQ ID NO: 3) | 94 ° C, 5 minutes 94 ° C, 20 seconds 27 cycles 55 ° C, 30 seconds 72 ° C, 30 seconds 72 ° C, 7 minutes | |
GAPDH-1D | 5'-catgagaccagcttgacgaa-3 '(SEQ ID NO .: 4) | |||
IFN | IFN-1U | 5'-tacagccaggtgtcaaatgc-3 '(SEQ ID NO: 5) | 94 ° C, 5 minutes 94 ° C, 20 seconds 29 cycles 59 ° C, 30 seconds 72 ° C, 30 seconds 72 ° C, 7 minutes | |
IFN-1D | 5'-tggaatcctcctcaaacagg-3 '(SEQ ID NO .: 6) | |||
Mx | floMx-F | 5'-aacagccaaggcaaagattg-3 '(SEQ ID NO .: 7) | 94 ° C, 5 minutes 94 ° C, 20 seconds 29 cycles 59 ° C, 30 seconds 72 ° C, 30 seconds 72 ° C, 7 minutes | |
floMx-R | 5'-aatgtccagctcctccttca-3 '(SEQ ID NO .: 8) | |||
ISG-15 | ISG-15-F | 5'-ctccatgtaatctgcagcaa-3 '(SEQ ID NO .: 9) | 94 ° C, 5 minutes 94 ° C, 20 seconds 26 cycles 57 ° C, 30 seconds 72 ° C, 30 seconds 72 ° C, 7 minutes | |
ISG-15-R | 5'-cagatctagtgcaggtgtga-3 '(SEQ ID NO .: 10) |
RT-PCR 결과를 도 11에 나타내었다. 도 11의 a는 고온 실험군을, b는 저온 실험군을, c는 대조군(PBS 처리군)을 나타낸다. 또한 a 및 b에서 레인 1 및 2는 백신침지군이며, 레인 3 및 4는 백신주사군이고, c에서 레인 1 및 2는 음성 대조군이고, 레인 3 및 4는 PBS 투여군이다.RT-PCR results are shown in FIG. 11. 11 a shows a high temperature experimental group, b a low temperature experimental group, and c represents a control group (PBS treatment group). In addition, lanes 1 and 2 in the a and b are immersion groups, lanes 3 and 4 are the vaccine injection groups, lanes 1 and 2 in the c are negative controls, and lanes 3 and 4 are the PBS administration groups.
상기 결과는 고온 및 저온 처리된 백신을 접종한 실험군에서 대조군에 비해 면역관련 유전자의 발현량이 증가하였음을 보여준다. 특히 고온 처리된 백신을 사용한 실험군에서의 면역관련 유전자의 발현량이 저온 처리된 백신을 사용한 실험군에서의 면역관련 유전자의 발현량에 비해 더 높은 것으로 보아 백신을 고온 처리하는 것이 유리한 것으로 확인되었다. The results show that the expression level of the immune-related genes was increased in the experimental group vaccinated with the hot and cold treatment compared to the control group. In particular, since the expression level of the immune-related genes in the experimental group using the high-temperature treated vaccine was higher than the expression level of the immune-related genes in the experimental group using the low-temperature treated vaccine, it was confirmed that the vaccine was treated with high temperature.
한편, 누적 폐사율 결과를 도 12에 나타내었다. 상기 도에서 보는 바와 같이, 대조구의 폐사율은 77%인 반면, 고온/백신주사군은 17%, 고온/백신침지군은 71%, 저온/백신주사군은 45%, 저온/백신침지군은 50%의 폐사율을 나타내었다. 상기 결과로부터 저온보다는 고온에서의 처리가 효과적이며 침지보다는 주사방법이 효과적임을 알 수 있었다. 고온/백신주사군의 경우 상대생존율이 78%로 가장 높아 가장 효과적인 것으로 나타났다. Meanwhile, the cumulative mortality results are shown in FIG. 12. As shown in the figure, the mortality of the control was 77%, while the hot / vaccine injection group was 17%, the hot / vaccine injection group was 71%, the cold / vaccine injection group was 45%, and the cold / vaccine immersion group was 50%. % Mortality was shown. From the above results, it can be seen that the treatment at a higher temperature than the low temperature is more effective and the scanning method is more effective than the dipping. The high survival / vaccine injection group showed the highest relative survival rate of 78%, which was the most effective.
실시예 7: 백신의 효능 평가Example 7: Evaluation of efficacy of vaccine
VHSV 백신의 효능 변화를 살펴보기 위하여, 상기 백신을 각각 20마리의 넙치(평균체중 41g, 17cm)에 접종하였다. 상기 접종 6주 후에, VHS 바이러스를 105 TCID50/마리(0.1ml/마리)로 주사한 다음, 대조군(PBS 투여)에 대한 폐사율, 라이소자임 활성 및 ELISA에 의한 에드와드균에 대한 응집항체가를 조사하였다. To examine the efficacy change of the VHSV vaccine, the vaccine was inoculated on 20 flounder (average body weight 41g, 17cm), respectively. Six weeks after the inoculation, the VHS virus was injected with 10 5 TCID 50 / mice (0.1 ml / horse), followed by mortality against the control group (PBS administration), lysozyme activity, and aggregated antibody titers against Edward bacteria by ELISA. Investigate.
라이소자임 활성 및 응집항체가는 하기 방법에 의해 수행하였다.Lysozyme activity and aggregated antibody titer were performed by the following method.
라이소자임 활성 측정Lysozyme Activity Measurement
1. 32 mM KH2PO4 0.435g를 3차 증류수 100 ml에 용해시켰다(이하 용액A).1. 0.435 g of 32 mM KH 2 PO 4 was dissolved in 100 ml of tertiary distilled water (hereinafter Solution A).
2. 32 mM K2HPO4 0.278g를 3차 증류수 50 ml에 용해시켰다(이하 용액B).2. 0.278 g of 32 mM K 2 HPO 4 was dissolved in 50 ml of tertiary distilled water (hereinafter Solution B).
3. 용액A 100 ml와 용액B 25 ml를 혼합하였다(이하 LY용액).3. 100 ml of solution A and 25 ml of solution B were mixed (hereinafter LY solution).
4. 마이크로코커스 리소데익티쿠스(Micrococcus lysodeikticus; Sigma no.M-3370)를 0.0014g를 LY용액 1ml에 녹였다(이하 균액).4. 0.0014 g of Micrococcus lysodeikticus (Sigma no.M-3370) was dissolved in 1 ml of LY solution (hereinafter, referred to as bacterial solution).
5. 96웰 평바닥 플레이트의 웰에 실험 혈청을 50 ㎕씩 3반복하여 분주하고, 여기에 균액을 50 ㎕씩 분주하였다.5. 50 µl of the test serum was repeatedly dispensed into three wells of a 96 well flat bottom plate, and 50 µl of the bacterial solution was dispensed thereto.
7. ELISA 리더를 이용하여 550 nm에서 0분 및 15분에 흡광도를 측정하였다.7. Absorbance was measured at 0 and 15 minutes at 550 nm using an ELISA reader.
8. 식 {(0분 흡광도값)-(15분 흡광도값)}×10000으로 계산하여 활성을 측정하였다. 8. The activity was measured by calculating the formula {(0 min absorbance value)-(15 min absorbance value)} × 10000.
응집항체가 측정Aggregated antibody measured
1. 96웰 둥근바닥 플레이트의 웰에 멀티 피펫을 이용하여 1×PBS를 25 ㎕씩 분주하였다. 1. Into a well of a 96 well round bottom plate, 25 μl of 1 × PBS was dispensed using a multi pipette.
2. 실험할 혈청을 첫 번째 열의 웰에만 25 ㎕씩 분주하였다. 2. The serum to be tested was dispensed 25 μl into the wells of the first row only.
3. 멀티피펫을 이용하여 첫 번째 열의 웰에서 11번째 열의 웰까지 25 ㎕씩 단계희석하였다.3. Dilute 25 μl from the first column wells to the 11th column wells using a multipipette.
4. 멀티피펫을 이용하여 FKC균을 12번째 열부터 첫 번째 열까지 순서대로 25㎕씩 분주하였다.4. Using a multipipette, 25 μl of FKC bacteria were sequentially dispensed from the 12th column to the first column.
5. 플레이트 뚜껑을 닫은 후, 안에 내용물이 잘 섞이도록 조심스럽게 흔들어준 다음, 냉장 상태로 하룻밤동안 보관하였다. 5. Close the plate lid, shake carefully to mix the contents inside, and store overnight in the cold.
상기 실험결과를 도 13에 나타내었다. 도 13a에서 보는 바와 같이, PBS 대조군의 폐사율은 90%인 반면, 바이러스 단독백신의 폐사율은 20%로, 상대생존율은 77%로 좋은 효과를 가졌다. 또한 도 13b 및 13c에서 보는 바와 같이, 라이소자임 활성 및 ELISA는 대조군에 비해 높은 활성을 나타냄을 확인할 수 있었다. The experimental results are shown in FIG. 13. As shown in FIG. 13A, the mortality rate of the PBS control group was 90%, whereas the virus alone had a mortality rate of 20% and a relative survival rate of 77%. In addition, as shown in Figure 13b and 13c, it was confirmed that lysozyme activity and ELISA showed a higher activity than the control.
실시예 8: VHSV 백신의 안전성 조사Example 8: Safety Investigation of VHSV Vaccine
본 발명의 백신의 안전성을 살펴보기 위하여, 넙치에 VHSV 백신을 투여한 후 여러 가지 장기(간, 비장, 신장, 심장, 위, 아가미)를 H&E 염색한 후, 현미경(×200)을 통해 PBS 투여군과 비교하였다. In order to examine the safety of the vaccine of the present invention, after administering the VHSV vaccine to the olive flounder H & E stains of various organs (liver, spleen, kidney, heart, stomach, gills), PBS group through a microscope (× 200) Compared with.
구체적으로, 10% BNF에 고정한 각 장기를 48시간 이내에 같은 고정액에 세절하여 재고정시켰다. 24시간 후 6-12시간 동안 흐르는 물에 수세한 후 농도별 알콜(70-100%)로 탈수시켰다. 이후, 자일렌으로 투명화하고 파라핀 침투시켜 파라핀 블록을 만들고 마이크로톰을 이용하여 세절(4 um)한 후 슬라이드에 부착시켰다. 상기 부착된 슬라이드를 50℃에서 하룻밤동안 건조시킨 후 H & E 염색법에 의해 염색시켜 현미경으로 관찰하였다.Specifically, each organ fixed in 10% BNF was cut and re-fixed in the same fixation solution within 48 hours. After 24 hours, the resultant was washed with running water for 6-12 hours and dehydrated with alcohol (70-100%) by concentration. Thereafter, the mixture was cleared with xylene and infiltrated with paraffin to make a paraffin block, and then cut into small portions (4 um) using a microtome and then attached to the slide. The attached slides were dried at 50 ° C. overnight and then stained by H & E staining and observed under a microscope.
상기 실험결과를 도 14에 나타내었다. 도 14는 VHSV 백신 처리 후, 간, 비장, 신장, 심장, 위 및 아가미의 현미경 사진을 보여준다. 상기 도에서 보는 바와 같이, VHSV 백신 투여시 모든 장기에서 정상 소견이 관찰되어 본 발명의 VHSV 백신은 넙치의 안전에 영향을 미치지 않는 것으로 나타났다.The experimental results are shown in FIG. 14. 14 shows micrographs of liver, spleen, kidney, heart, stomach and gills after VHSV vaccine treatment. As shown in the figure, normal findings were observed in all organs when the VHSV vaccine was administered, indicating that the VHSV vaccine of the present invention did not affect the safety of the flounder.
본 발명에 따른 백신은 넙치에서 발생하는 바이러스성 출혈성 패혈증을 효과적으로 예방할 수 있으므로, 넙치의 양식 산업 등에 유용하게 사용될 수 있다.Since the vaccine according to the present invention can effectively prevent viral hemorrhagic sepsis occurring in the flounder, it can be usefully used for the aquaculture industry of the flounder.
Claims (6)
- 불활성화된 바이러스성 출혈성 패혈증 바이러스 FP-VHS2010-01(KCTC 12153BP) 항원을 포함하는 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신.Inactivated viral hemorrhagic sepsis virus Inactivated vaccine for preventing viral hemorrhagic sepsis of olive flounder containing FP-VHS2010-01 (KCTC 12153BP) antigen.
- 제1항에 있어서, 상기 백신이 바이러스성 출혈성 패혈증 바이러스 FP-VHS2010-01을 18~22℃의 온도에서 배양시켜 수득되는 것을 특징으로 하는, 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신.The inactivated vaccine for preventing viral hemorrhagic sepsis of halibut according to claim 1, wherein the vaccine is obtained by culturing viral hemorrhagic sepsis virus FP-VHS2010-01 at a temperature of 18 to 22 ° C.
- 제1항에 있어서, 상기 백신이 바이러스성 출혈성 패혈증 바이러스 FP-VHS2010-01을 열 불활성화(heat inactivation)시키는 것을 특징으로 하는, 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신.The inactivated vaccine for preventing viral hemorrhagic sepsis of halibut according to claim 1, characterized in that the vaccine heat inactivation of the viral hemorrhagic sepsis virus FP-VHS2010-01.
- 제1항에 있어서, 상기 백신이 주사에 의해 넙치에 투여되는 것을 특징으로 하는, 넙치의 바이러스성 출혈성 패혈증 예방용 불활성화 백신.The inactivated vaccine for preventing viral hemorrhagic sepsis of the flounder, according to claim 1, wherein the vaccine is administered to the flounder by injection.
- 제1항 내지 제4항 중 어느 한 항에 따른 백신을 어류에 투여하는 것을 포함하는, 넙치의 바이러스성 출혈성 패혈증의 예방 방법.A method for preventing viral hemorrhagic sepsis of a flatfish, comprising administering the vaccine according to any one of claims 1 to 4 to fish.
- 제5항에 있어서, 상기 백신을 어류의 복강에 주사하는 것을 특징으로 하는 넙치의 바이러스성 출혈성 패혈증의 예방 방법.The method of preventing viral hemorrhagic sepsis of flounder, according to claim 5, wherein the vaccine is injected into the abdominal cavity of the fish.
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KR101768320B1 (en) | 2015-09-11 | 2017-08-30 | 제주대학교 산학협력단 | Method for concentration of VHSV using 2-step ultrafiltration |
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