KR101677947B1 - A vaccine for prevention of miamineis aivus infection for fish - Google Patents

Abstract

The present invention relates to a method for cultivating a scuticaccharide; Adding formalin to the culture and reacting the culture, and obtaining a scuticacid inactivated liquid from the formalin-added culture.

Description

{A VACCINE FOR PREVENTION OF MIAMINEIS AIVUS INFECTION FOR FISH}

The present invention relates to a vaccine for preventing flukasquitica.

Flounder is an important breed that accounts for about 52% of Korean fish production, but it causes many damages by infectious disease every year. The results of the survey on the occurrence of diseases in the flounder farms in Jeju Island and Wando Island, which are the major flounder producing regions, showed an average mortality rate of 27 ~ 30%, and the main diseases were scuticchaemia, streptococcal disease, VHS, Edwardian disease and Vibrio disease.

Scuticaria is one of the most troublesome diseases that occur in flounder, and it is a parasitic disease with high economic losses due to high mortality and no treatment measures. Sututika sac is parasitic on the gills, the body surface and the fins, and the tissue collapses. When the symptoms progress, the muscle is exposed and penetrates into the brain and various internal organs, causing necrosis of the tissue.

There are many kinds of Sutuca cordata, but in Korea, only Miamiensis avidus is known as a pathogenic organism having strong pathogenicity in flounder.

Scuticaria is a parasitic disease caused by intestinal and parasitic infestation of scuticociliates in the muscles and brain tissues including the body surface and gills of many marine aquaculture species, especially infected with flounder causing massive deaths, Sea bass, black sea bream, rockfish, sea bass, and blowfish (Korean Patent Publication No. 10-2013-0078902). This infectious disease occurred throughout the year and continued to exhibit cumulative mortality, and the epidemic epidemic was epidemic in autumn and early spring, when water temperature started to fall, but it was recently reported to occur throughout the year. In the sea bass, ducks, rocks and bushes that are cultivated in the aquaculture or festive ceremony, the scutica is mainly parasitic and fins, which break down the fins and roughen the surface of the body. It mainly causes secondary infections (Vibrio or Slug bacteria) Most of them lead fish to our company.

The present inventors completed the present invention by studying a vaccine to prevent the damage of Sukutika crabs in fishes such as flounder.

It is an object of the present invention to provide a vaccine for the prevention of a scuticacid infection.

In order to achieve the above object,

Culturing the sutucca insect;

Adding and reacting formalin to the culture; and

And obtaining a scuticacid inactivated liquid from the formalin-added culture

The present invention provides a method for producing a fish vaccine against Sucutika spp.

The vaccine prepared by the production method of the present invention has an effect of preventing the infection against Sukutika infestation and lowering the mortality of fish when infected with Sukutika infestation.

Figure 1 shows Western blotting results for homologous antiserum of serotype type I (lane 1,2,3,4,5,8) and type II strains (lane6,7).
M, protein marker; N, negative control; 1, strain PH12-A1; 2, strain PH12-A2; 3, strain PH12-B1; 4, strain PH12-B2; 5, strain JJ12-A1; 6, strain JJ12-A2; 7, strain JJ12-A3; 8, strain JJ12-B1
FIG. 2 shows the result of Immobilization assay using anti-scuticum rabbit serum.
Figure 3 shows the cumulative mortality due to low and high concentrations of Sukutika pufferfish infections.
Fig. 4 shows the symptoms of lung disease caused by infection with scutica.
Fig. 5 is a histopathological examination result of flounder tissues of each concentration at the 1st week of inoculation with scutica.
Figure 6 shows the results of recombinant protein expression from the flounder immunoglobulins TNF-α (A, 26 kDa), CD40 (B, 33 kDa), IL-1β (C, 30 kDa) and G- .
M, marker; Lane 1, 0 mM IPTG, Supernatant; Lane 2, 0.1 mM IPTG, Supernatant; Lane 3, 0.5 mM IPTG, Supernatant; Lane 4, 1.0 mM IPTG, Supernatant; Lane 5, 0 mM IPTG, Pellet; Lane 6, 0.1 mM IPTG, Pellet; Lane 7, 0.5 mM IPTG, Pellet; Lane 8, 1.0 mM IPTG, Pellet.
Figure 7 shows the pET22b. 6H. MBP. G-CSF expression analysis (about 65 kDa). M, marker; 1, Uninduction Total; 2, 37 캜 - Induction Total; 3, 37 캜 - Induction insoluble; 4, 37 캜 - Induction soluble; 5, 25 캜 - Induction Total; 6, 25 캜 - Induction insoluble; 7, 25 ° C -Induction soluble.
FIG. 8 shows the results of quantitative real-time PCR-induced flounder immunity-related genes (1, 3, 6, 12 and 24 hours) after stimulation of rPoGCSF in flounder leucocytes . (A) G-CSF; (B) TNF; (C) TLR2; (D) TLR3; (E) IL-1 [beta]; (F) IL-6; (G) NKEF
FIG. 9 shows the effect of G-CSF recombinant protein treated by pH on the expression of IL-1β and TNF in flounder leukocytes. (A) IL-1?; (B) TNF
Figure 10 shows the effect of G-CSF recombinant protein treated by temperature on IL-1 [beta] and TNF expression in flounder leukocytes. (A) IL-1?; (B) TNF
Figures 11 to 13 show the results of histological examination after administration of the vaccine.
14 shows a scuticacid agglutination reaction (A: negative control, B: agglutination)
Figure 15 shows cumulative mortality for anthrax infections after vaccination.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. However, it should be understood that the present invention is not limited to the embodiments disclosed herein but may be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

≪ Example 1 >

<1-1> Sukutika separation

Sucutica spp. Was isolated from the brain of a flounder showing the presence of scuticum in fourteen flounder farms in Pohang, Jeju and Wando, and cloned 5 times by limiting dilution method using a CHSE-214 cell-line distributed at 96 wells Pure separation.

<1- 2> Sukutika Charismatic

The isolated strain was cultured in CHSE-214 cell-line, centrifuged at 500 g for 5 min, suspended in 100 μl TE buffer, and DNA was isolated using DNA extraction kit (Bioneer).

For 18S rDNA sequence analysis, PCR was performed using a universal primer set (Table 1). After DNA amplification, target DNA was cut out and purified using a DNA gel extraction kit (Millipore). The purified DNA was cloned using T-blunt PCR cloning kit (Solgent) and sequenced.

Primer sets used for gene sequence analysis

As a result of isolating 52 strains of Sutucica from 14 farms in Jeju, Pohang and Wando, all strains were 100% identical to 18S rDNA (EU831206) of 1759bp of Miamiensis avidus (Table 2).

All of the 52 strains isolated in the present invention were identified as M. avidus, confirming that M. avidus was the major race causing scuticacidosis in cultured flounder in Korea.

a 18S rDNA sequence identity compared to a Miamiensis avidus (EU831206)

b Immobilization activities of anti-PH12-A1 strain sera prepared in rabbits.

c Jung S.J., E.Y. Im, M.C. Struder-Kypke, S.I. Kitamura, P.T.K. Woo, 2011. Small subunit ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1 gene sequences of 21 strains of the parasitic scuticociliate Miamiensis avidus (Ciliophora, Scuticociliatia). Parasitol Res. 108: 1153-1161. &Lt; RTI ID = 0.0 &gt;

&Lt; Example 2 >

 <2-1> Production of rabbit antiserum

Antigen protein analysis, immobilization assay and cox I gene analysis were performed to investigate the serotype of 52 strains of M. avidus isolated in this study. For immunoassay, strain PH12-A1 of strain No. 1 and strain PH12-A1 of strain No.1 were used. 6 strain StJJ12-A2 was immunized with rabbit anti-scutica rabbit serum.

 &Lt; 2-2 > Antigen protein analysis

Separated Sututika pellet was cultured in CHSE-214 cell-line, and then 3 ml was taken, centrifuged at 500 g for 5 min, washed 3 times in L-15, and finally suspended in 50 μl TE buffer. The suspension of Sucutica spp. Was repeatedly subjected to freezing-thawing three times at -80 ° C to break down the cells of Sucutica spp., Mixed with 1: 1 in 5x sample buffer, and thermally denatured at 100 ° C for 10 minutes. After heat denaturation, protein pattern was confirmed by SDS-PAGE using 12% gel. In addition, the proteins separated by SDS-PAGE were stained with anti-rabbit IgG (whole protein), anti-rabbit IgG (anti-rabbit IgG) Alkaline Phospatase conjugated goat antibody) and developed with BCIP® / NBT solution.

Antigenic protein analysis of the rabbit antiserum showed that the strains were divided into two types. Type II strains reacting with antisera of anti-type II strain have approximately 30 kDa antigen protein and type I stains reactive with anti-type I stain have approximately 20 kDa antigen protein. Figure 1 shows the results of antigen protein detection for several strains (M, protein marker; N, negative control; 1, strain PH12-A1; 2, strain PH12-A2; 3, strain PH12- 6, strain JJ12-A2; 7, strain JJ12-A3; 8, strain JJ12-B1) and the corresponding types of antigen protein by strain are shown in Table 2.

<2-3> Immunoassay

The anti-scutica rabbit serum (anti-PH12-A1 or anti JJ12-A2) was diluted 10, 20, 40, 80, 160, 320, well. In other words, the step-diluted antibody and scuticacid were reacted at 20 ° C for 1 hour and then immobilized or agglutinated by a microscope.

Immunization test of 52 strains against two rabbit antiserum (anti-PH12-A1 rabbit serum and anti-JJ12-A2 rabbit serum) and anti-PH12-A1 rabbit serum All strains showing the same antigenic protein pattern were aggregated or inactivated at 320-1280 fold (Fig. 2). However, some strains did not react with this antibody and showed vigorous movement and different serotypes. These results were consistent with the results of antigen protein analysis.

<2-4> Cox I gene sequence analysis

Cox I gene has been used as a marker gene to classify animal species and recently it has been used in species diversity research in the ciliate gate. Therefore, cox I gene was searched for serum type of this study, The intergenic genotypes were analyzed. The isolated strains were isolated using DNA extraction kit (Bioneer) and sequenced after PCR by PCR according to Jung et al. (2011) method (Table 1).

Genetic analysis of the cox I gene used to investigate the species diversity of ciliates showed that 100 genes of the 50 strains considered to be of the same serotype were 100% identical to those of the type II EU831214). In addition, two strains (strains No. 6 and 7) with different serotypes in this study were consistent with type I (EU831221) classified by Jung et al., (2011).

Thus, as in EU831214, the 50 strains of type II have the cox I gene of SEQ ID NO: 6 below and the two strains of type I have the cox II gene of SEQ ID NO: 7 as in EU831221 (Table 3).

&Lt; Example 3 >

<3- 1> Selection of strain for pathogenic experiment

All strains isolated from Jeju, Pohang and Wando were the same species as Miamiensis avidus. Two serotypes were detected from serotype analysis. Therefore, in this study, therefore, five strains (strain No. 1: PH12-A1, strain No. 6: JJ12-A2, strain No. 7: JJ12-A3, strain No. 5; 8: JJ12-B1, strain No. 33: WD13-A1) was selected and used for pathogenic investigation.

<3- 2> Preparation of Flounder and Sututika

For pathogenic experiments, the flounder (average 12g, 11.7cm) was distributed from the National Institute of Fisheries Science and Breeding Research Center to the fish breeding room. Experimental specimens were weighed in a 0.5-ton fiber-reinforced plastic (FRP) water bath for 2 weeks at 20 ° C and used for experiments after confirming that they did not infect major flounder diseases (VHSV, RSIV, VNNV, Scutica, etc.).

Five strains of Sucutica spp. For use in infection experiments were cultured on a CHSE-214 cell-line in a 75 cm2 cell culture flask for large-scale culture, then inoculated with four squid flasks per strain and incubated for 7 days at 20 ° C Lt; / RTI &gt; Sututika sacrificed for the infection experiment was centrifuged at 500 g for 5 minutes, collected, and washed three times in Leibovitz's L-15 Medium (L-15) and counted using a cell-counting chamber.

 &Lt; 3-3 >

In the pathogenic experiment, 15 liters of flounder were housed in a 20L water tank and then infected by artificial infection. The infectious concentrations were divided into two groups and injected at a high concentration (10 ⁵ cells / 100 μl / fish) and low concentration (10 ⁴ cells / 100 μl / fish) for each strain. The control was L-15 at 100 μl / Respectively. After incubation at 19 ~ 20 ℃, the virus was recovered 5L per day. At the time of death, the dead lungs were removed and the Sucutica spp. Was observed under the microscope in the brain and the arm.

In the case of the experimental group inoculated at a high concentration (10 ⁵ cells / fish), mortality began to occur from the third day of infection together with severe multiple symptoms (FIG. 3 (b)). 33 strains showed 100% mortality on the 8th day of infection. Experimental groups of other strains also showed 93.3 ~ 100% mortality rate during the experimental period (28 days).

On the other hand, in the low concentration experiment (10 ⁴ cells / fish), death occurred from the 7th day of infection and 53.3 ~ 93.3% of deaths occurred during the experimental period (28 days) 3 (a)). It was confirmed from the brain of the dead fish and the gill, and it was confirmed that the virus was caused by Sukutika infection.

All strains were associated with high mortality regardless of the serotype of the infected samples. However, the serotype I strains showed lower pathogenicity than the type II strains. The symptoms of the dead fish according to concentration are shown in Fig.

&Lt; Example 4 >

All isolates were identified as M. avidus, and two types of serotype were present. Pathogenic investigations showed that all five strains selected from the two serotypes were highly pathogenic.

One strain with high virulence in two serotypes was selected for vaccine strain and used for vaccine development (Table 4).

Vaccine candidate strains selected from serotype characteristics and virulence studies

<Example 5> Survey on efficacy and safety of Sucutika vaccine

<5-1> Production of vaccine for inactivating scutica

Two types of serotypes were present in Sututika spp. The representative strains shown in Table 45 were cultured and then inactivated with 0.3% formalin to prepare Sutucica spp. Vaccine. The detailed method is as follows.

① Remove the cell of T-75 flask (falcon) in the same way as the cell culture, transfer the whole amount to 850 Roller bottle (corning), and put the medium up to 200ml line.

② Cultivate at 0.25 rpm, 20 ℃ for about 50 days in a convection incubator.

③ After culturing the cells, add 1 ml of Scutica and cultivate at 0.25 rpm, 20 ℃ for 8 days in a convection incubator.

④ Transfer all the scutica cultures to a beaker and agitate and treat the formalin with 0.3%. (When the formalin treatment is done, put the beaker in the ice and slowly put it in one drop.)

⑤ After reacting for 12 hours in a refrigerator at 4 ℃, it is checked whether or not it is inactivated. When all individuals are inactivated, remove the supernatant by pipette about half.

⑥ After removing the supernatant liquid, dilute the diluted Sucutica solution in a 50 ml tube and centrifuge (500xG, 20 min, 4 ℃).

⑦ Discard the supernatant and wash 3 times with L-15 (500xG, 20min, 4 ℃).

⑧ Collect the washed scutica into one tube, and add L-15 so that the total volume becomes 100 ml.

⑨ Count the scutica using a hemocytometer and adjust it to the desired concentration by dilution or concentration.

⑩ Inoculate 0.1 ml of vaccine into the flounder.

The hemocytometer was calculated according to the following formula (1).

<Formula 1>

<5-2> Investigation of efficacy according to the concentration of Sukutika puffer vaccine

 The efficacy of Sucutika vaccine was investigated by intraperitoneal injections of Sucutika inactivated vaccine alone or in various concentrations and injected into flounder (15.4g) at 3 weeks of injection. Relative survival rates were examined (Table 6).

<Formula 2>

Relative survival rate = (1- vaccine cumulative mortality / cumulative cumulative mortality) 100

Effectiveness of vaccine administration concentration

The efficacy of each vaccine was determined by comparing the relative survival rates of the strains of type I (strain7) and type II (strain 31) at high concentration (6.6x10 ⁵ cells / fish) and low concentration (6.6x10 ⁴ cells / fish) . There was no difference in efficacy between Type I (strain 7) and type II (strain 31) vaccines, so a low-dose vaccine (6.6x10 ⁴ cells / fish) was considered economically effective (Table 6).

Mix (type I and type II) vaccines, a mixture of Type Ⅰ and Ⅱ, showed no effect on concentration and showed a protective effect against both serotypes.

Relative survival rate for anthropogenic infection by vaccine dose

<5-3> Cross-vaccine efficacy of serotype

The serotype cross-vaccine efficacy was obtained by inoculating the flounder (9 cm, 7 g) with the type I and type II (10 ⁴ cells / fish) The relative survival rate after homologous strain and heterologous strain of each vaccine after attack infection (5x10 ⁴ cells / fish) was examined one week after the second vaccination.

The cross - vaccine efficacy of the serotypes was investigated by homologous strain and heterologous strain after the vaccination of type Ⅰ and type Ⅱ strains. Relative survival rates for homologous strains were as high as 75% and 80%, respectively (Table 7).

 Relative survival rates for heterologous strains were 58% and 60%, respectively, lower than the relative survival rate of homologous pathogens, but higher than 50% for heterologous pathogens (Table 8).

Relative survival rate for anthropogenic infection after cross-over vaccine serotype

<5-4> Safety investigation of Sututika puffer vaccine

In this study, we investigated the effects of type II (strain 31) vaccine on the experimental flounder (21 g), which were inoculated with 10 ⁶ cells / fish (High), 10 ⁵ cells / fish (Medium) and 10 ⁴ cells / And its safety and pathological findings were confirmed. Glucose, ALT, AST and total protein concentrations were measured using an automatic hematology analyzer. Histological examinations were performed according to the commercial method after fixing the kidney, spleen, liver, and gill in neutral formalin.

The safety of Sututika vaccine was evaluated by biochemical features and histopathological examination after vaccination. The blood biochemical characteristics (AST, ALT, GLU, and TP) were not significantly different between the groups, and the concentration was considered to be safe for flounder (p> 0.05, Table 9).

Changes in hematological characteristics (Mean ± SD)

Histopathological examination after vaccination with scutica showed no difference in pathological histology of the liver, kidney, spleen and intestine of the vaccine or control, indicating that the scutica vaccine was safe for flounder (Fig. 5).

<Example 6> Effectiveness of adjuvant-added scuticacid vaccine

The efficacy and safety of adjuvant - containing vaccine were investigated for the enhancement of vaccine efficacy and duration. In this study, molecular adjuvant and compound adjuvant, which are immune genes of flounder,

<6-1> Preparation of Molecular Adjuvant

Cloning and recombinant protein production of halibut immune-related genes

TNF-α, G-CSF, IL-1β, and CD40 were selected as candidates for the olive flounder immune-related genes to produce molecular adjuvant from olive flounder. Information on these genes was obtained from the GenBank database of the National Center for Biotechnology Information The genes already registered were selected and used in this study (Table 10). To amplify the ORF of each gene, primers were prepared by adding EcoR I to 5 'and Xho I base to 3' (Table 11). The primers were amplified by PCR, and then the immune genes were extracted by gel extraction and cloned into pET 22b vector. After cloning, the mutation of each gene and amino acid sequence was confirmed by sequencing. The pET 22b vector inserted with each of the flounder immune genes was transformed into E. coli BL21 (DE3). IPTG (isopropyl-D-thiogalactoside) prepared at 0, 0.1, and 0.5 mM was added to transformed E. coli BL21 and cultured at 37 ° C for 4 hours to induce the expression of the recombinant protein of the flounder immune system.

TNF (Tumor Necrosis factor) -a is a 17 kDa protein that is synthesized from a variety of cells stimulated by endotoxin, inflammatory mediators, or cytokines such as IL-1. Various TNF superfamilies have been identified in flounder and have been reported to function similar to TNF-α reported in mammals through their molecular biology and expression analysis. In the present invention, TNF of the registered number BAA94969 of GenBank was used (SEQ ID NO: 8).

CD40 (Cluster of Differentiation 40) is a member of the tumor necrosis factor (TNFR) superfamily and is a member of the B cells, dendritic cells, endothelial cells, fibroblasts, Epithelial cells, and the like. CD40 is also a typical antigen receptor that causes an immune response. In the present invention, CD40 of the registered number BAC87848 of GenBank was used (SEQ ID NO: 9).

IL-1β (Interleukin 1β) is a typical cytokine that causes an inflammatory response. Especially, in flounder, their functions have been analyzed by various researchers and the ability of IL-1β to induce immune response has been analyzed through cDNA microarray technique. In the present invention, IL-1? Of the GenBank registration number BAB86882 was used (SEQ ID NO: 10).

The granulocyte colony stimulating factor (G-CSF) is a glycoprotein cytokine that plays an important role in the hematopoiesis of phagocytic neutrophils and plays a pivotal role in neutrophil-based immune responses. Their presence was also reported in fish. In the present invention, G-CSF of the registered number BAE16320 of GenBank was used (SEQ ID NO: 11).

In the present invention, a recombinant protein was designed to investigate the possibility and utility of TNF-α, CD40, IL-1β and G-CSF, which are important immunity-related genes in flounder, as molecular adjuvant. However, SDS-PAGE analysis showed that only TNF-α (about 26 kDa), CD40 (about 33 kDa), IL-1β (about 30 kDa) and G-CSF Was expressed in insoluble form (Fig. 6).

The nucleotide sequence of the oligonucleotide primer

Soluble form recombinant protein production and purification

G-CSF was selected from four candidates for producing the flounder immunity-related recombinant protein and cloned by attaching MBP (Maltose binding protein, 40 kDa) to the N-terminus of G-CSF as a fusion partner and adding IPTG to the recombinant protein . Expression-induced G-CSF recombinant proteins were purified using MBP affinity resin.

 In order to express G-CSF in E. coli BL21 (DE) stably in a soluble form, MBP was attached to the N-terminus of G-CSF as a fusion partner, Cloning was performed, and it was confirmed that soluble form of recombinant protein (rPoGCSF) was produced at 25 ° C by IPTG induction. In particular, rPoGCSF was expressed in E. coli BL21 (DE), and the purified protein was purified using MBP affinity resin and eluted with elution buffer containing 10 mM maltose (Fig. 7) .

<6-2> In vitro stimulation of leukocyte immune gene by molecular adjuvant

G- CSF Of the recombinant protein (rPoGCSF)  Depending on concentration stimulus Leucocyte  Investigation of the expression level of immune-related genes

  Fluconazole (rPoGCSF) was added at concentrations of 10, 1, and 0.1 μg / mL for 1, 3, 6, 12 and 24 hours after peritoneal flukes were isolated from peritoneal flounder. Total RNA was isolated from stimulated white blood cells by using RNA purification kit (Amersham Pharmacia, Piscataway, NJ, USA). To extract cDNA, the extracted total RNA was purified using Recombinant DNase I-RNase free kit (TaKaRa, Japan) And treated with DNase according to the manufacturer's method. After that, total RNA treated with DNase was synthesized with cDNA using cDNA synthesis kit (Stratagene, LaJolla, CA, USA).

  In order to investigate the expression level of the flounder immunity-related genes for the time stimulation, 1 μl of synthesized cDNA template, 1 μl of forward primer and reverse primer, 12.5 μl of SYBR Green I (TaKaRa, Japan) and 9.5 μl of PCR grade water After mixing to a total volume of 25 μl, quantitative real-time PCR was performed using a Thermal Cycler DICE Real-Time System (Takara). The expression level of rPoGSCF by concentration / time of stimulation was determined by using specific primers of G-CSF, TNF-α, NKEF, TLR2, TLR3, IL-1β and IL- Respectively. Specific primers for each gene used in this study are listed in Table 9.

1) G-CSF showed the highest expression level at 1 ㎍ / ㎖ and 12 hours. The concentration of G-CSF at 10, 1, and 0.1 ㎍ / The expression of G-CSF gene was increased (Fig. 8A).

② TNF-α: The expression level tended to increase with time, and the expression level tended to increase with concentration-dependent stimulation of 1 and 0.1 μg / ml (FIG. 8B).

③ TLR2: Expression amount tended to increase with 12 hours after stimulation (Fig. 8C).

The expression level of TLR3 was increased with time, and the expression level of 10, 1, and 0.1 ㎍ / ㎖ was increased with increasing concentration of TLR3 (Fig. 8D).

⑤ IL-1β: The expression level of IL-1β was increased at higher concentrations at 10, 1 and 0.1 ㎍ / ㎖ until 6 hours of stimulation, and the highest expression was observed at 12 hours (Fig. 8E).

⑥ IL-6: The higher the concentration, the higher the expression level and the highest expression was observed at 12 hours for 10, 1 and 0.1 ㎍ / ㎖ of stimulation until 6 hours of stimulation (Fig. 8F).

⑦ NKEF: The expression level tended to increase with time, and the higher the concentration of 10, 1, and 0.1 ㎍ / ㎖ of the stimulus, the higher the expression level. The GK CSF recombinant protein induced the NKEF gene (Fig. 8G).

rPoGCSF  Stability Studies-I: Expression of Immune-Related Gene by pH Treatment Induction ability  Research

<PH of rPoGCSF to be used as a molecular adjuvant>

To investigate the change of activity according to the condition, rPoGCSF (100 μg / mL) was used for the experiment after 24 hours exposure at pH 2, 4, 6, 8, 10 and 12. The expression levels of IL-1β and TNF were measured by quantitative real-time PCR after rPoGCSF exposed to 1, 3, 12, and 24 hours of flukescent white blood cells.

After rPoGCSF was treated at pH 2, 4, 6, 8, 10, and 12 for 24 hours, the level of IL-1β and TNF was examined by stimulating flounder leukocytes. The expression level of IL-1β was not different according to the pH condition, but increased with the treatment time, and all the experimental groups were significantly higher than the control group at 24 hours. In the pH 6 and pH 10 experimental groups, the highest expression levels were 32.3 and 32.7 times, respectively, at 24 hours after treatment (FIG. 9A). The amount of TNF expressed in the white blood cells of the flounder was similar to that of the control until 3 hours of stimulation but slightly increased at 12 hours. (P <0.05, Fig. 9B) at 24 hours after the stimulation,

  The expression levels of IL-1β and TNF were different in each stimulation time, but they were stable in a wide pH range as compared with the control without stimulation at all the pH conditions examined. It could be used as an adjuvant of oral vaccine because it can be highly active as an adjuvant considering the pH of flounder fluids and stable in acidity.

rPoGCSF  Stability study-II: Expression of immune related genes by temperature treatment Induction ability  Research

  RPoGCSF (100 μg / mL) was used for the experiment after exposure to rPoGCSF at 10, 20, 25, 30, 35 and 42 ° C for 24 hours to investigate the activity change of rPoGCSF to be used as a molecular adjuvant according to temperature condition. The expression levels of IL-1β and TNF were measured by quantitative real-time PCR after 1 hour, 3 hours, 12 hours, and 24 hours of reaction with flounder leukocytes in the same manner as stimulation by pH conditions in rPoGCSF exposed at each condition Respectively.

After rPoGCSF treatment at 10, 20, 25, 30, 35, and 42 ℃ for 24 hours, IL-1β and TNF expression levels were examined by stimulating neutrophil white blood cells. The expression level of IL-1β was significantly higher in all experimental groups than in the control group at 24 hours after treatment. Especially, at 20 and 25 ℃, the expression level of IL-1β was significantly higher than that of the control group at 24 hours (P < 0.05, Fig. 10A). TNF expression level was similar to that of control at 12 hours in all experimental groups, but significantly higher at 24 hours than control. In particular, the 25 ° C experimental group exhibited significantly higher expression than the 24 hour expression level of the other experimental groups (p <0.05, FIG. 10B).

IL-1β and TNF were expressed at different stimulation times. However, the expression level of IL-1β and TNF was higher than that of the untreated control at all the temperature conditions examined. It is considered that the high activity is high when used as an adjuvant.

Statistical processing

Statistical analysis was performed using the Duncan's multiple range test (Duncan, 1955) using the ANOVA-test. The mean significance was calculated using the SPSS Version 10 (SPSS, Michigan Avenue, Chicago, Respectively.

Example 7: Preparation of adjuvant-added scuticacid inactivated vaccine

<7-1> Preparation of compound adjuvant

In a previous study, compound adjuvants were selected for sepppic Gel 01, which was found to be safe in flounder.

<7-2> Preparation and vaccination of Adjuvant-added scuticacid inactivated vaccine

Type Ⅱ Formicin vaccine was prepared from squid fungus and mixed with compound adjuvant or molecular adjuvant. In previous studies, low-dose 6.6 × 10 ⁴ cells / fish) were tested for vaccine efficacy and mixed with low molecular weight molecular adjuvant or compound adjuvant. The experimental groups were as shown in Table 12 below.

Experimental Section for Adjuvant-Added Scuticacid Vaccine Efficacy and Safety Survey

<7-3> Investigation of safety of Adjuvant-added scuticacid vaccine

Blood biochemical properties

The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose and total protein were measured at 1, 2, and 3 weeks after vaccination. The blood biochemical profile after vaccination showed no difference between the vaccine and the control, and it was considered safe because the vaccine did not cause mortality (Table 13).

Changes in hematological characteristics (Mean ± SD)

Histopathological  inspection

After 1, 2, and 3 weeks of vaccination, liver, kidney, spleen, and bowel were fixed with 10% neutral formalin and stained with H & E staining.

There was no difference in the histopathological examination of the liver, kidney, spleen and intestine of the adjuvant-added vaccine, vaccine and control, and the scutica vaccine and the adjuvant-added scuticaca vaccine were judged safe for flounder (FIG. 11 to FIG. 13 ).

<7-4> Investigation of efficacy of Adjuvant-added scuticacid vaccine

Cohesion  Research

 In the coagulation test, no aggregation was observed before and during vaccination, but antibody titers were observed in vaccine, compound adjuvant, and molecular adjuvant. There was no difference between the vaccine groups (Table 14).

Aggregate Survey Results

Lysozyme  Activity investigation

From 3 hrs after vaccination, all vaccine groups showed higher activity than the control group, and V group was higher until 7th day. Vaccination has been shown to activate specific immune systems as well as nonspecific immune systems (Table 15).

Lysozyme activity investigation

Cumulative mortality rate due to anthropogenic infection

To investigate the efficacy of the vaccine, mortality was investigated after infection with S. aureus. After adjusting the concentration of the pure cultured Sutuca cordata to 5 × 10 ⁵ cells / ㎖, the mortality rate was monitored by intraperitoneal injection of 0.1 ml each.

In the control group, all the individuals died from the 12th day of the infection, but the vaccine group showed 37 ~ 53% cumulative mortality on the 17th day of the infection. Specifically, 37% of the vaccine-free vaccine, 43% of the compound adjuvant vaccine, and 53% of the molecular adjuvant vaccine vaccine did not contain the adjuvant (FIG. 15).

In the present study, vaccine without adjuvant showed high defense against Sucutica spp., And it was judged that adjuvant addition of Sucutica spp. Vaccine did not improve vaccine efficacy.

<110> NIFS <120> A VACCINE FOR PREVENTION OF MIAMINEIS AIVUS INFECTION FOR FISH <130> GNP 150121 <160> 35 <170> PatentIn version 3.2 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Forward primer for PCR of the 18S rDNA of Miamiensis avidus. <400> 1 acctggttga tcctgccagt 20 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse primer for PCR of the 18S rDNA of Miamiensis avidus. <400> 2 tgatccttct gcaggttcac ctac 24 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Internal primer for PCR of the 18S rDNA of Miamiensis avidus. <400> 3 ggcgcgtaaa ttacccaatc 20 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > Forward primer for PCR of the cox I of Miamiensis avidus. <400> 4 tcaggagctg cmttagchac yatg 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PCR of the cox I of Miamiensis avidus <400> 5 tartatagga tcmccwccat aagc 24 <210> 6 <211> 900 <212> DNA <213> Miamiensis avidus <400> 6 attagattag aattagctca tccaggaagt ccatttttta aaggagactc tttaagatat 60 ttacaagtta ttacagcaca tggtttaatt atggtattct ttgttgtagt tcccgttatt 120 tttggggctt ttgcaaattt tttaataccg taccatattg gttctaaaga tgtggcttac 180 cctagactaa atagtatagg tttttgaatt caaccttgtg gttttatttt agtatctaaa 240 atagcatttt taaggccaca atactgaaga tactacgata aagcttctta ctattttcct 300 ttacttgata aaagtaataa tagagcattt aatgaattta ataacactaa taacattttt 360 cagtttagag cattacaaag gtatgcttta gacgaacata cactattttg aaaacctaaa 420 ctaacaaata aatatataaa ttatgaaaat tattctggaa ttcctttaaa attattattt 480 tgaaaagata ttattaacta cccagaatct ttttgatacg ttgtaagtcg tattaataaa 540 gtacgtagaa aaaaagtata ttttactaaa tgttctaaca gaactttaac tacagctggt 600 tgaactttta ttacaccatt tgcatcaaat gtaaaatata caggtattgg tgctcaagat 660 ttactattag tatcagttgt ttttgctggt attagttcga cagtatcgtt cacaaattta 720 ttaattacaa gacgtacttt atgtatgcct ggtatgagac atagaagagt gctattacca 780 tttgtaacta taggtatttt ttttgcatta agaatgttgg ctattattac acctgtgtta 840 ggagcggcta tgattatgat ggctttagat agacattgac aaactacatt ttttgatttt 900                                                                          900 <210> 7 <211> 900 <212> DNA <213> Miamiensis avidus <400> 7 attagattag aattagctca tccaggaagt ccatttttta aaggagactc tttaagatat 60 ttacaagtta ttacagcaca tggtttaatt atggtatttt ttgttgtagt tcctgttatt 120 tttggggctt ttgcaaattt tttaataccg taccatattg gttctaaaga tgtggcttac 180 cctagactaa atagtatagg tttttgaatt caaccttgtg gttttatttt agtatctaaa 240 atagcatttt taaggccaca atactgaaga tactatgata aagcttctta ctattttcct 300 ttacttgata aaagtaataa tagaacattt aacgaattta ataacactaa taacattttt 360 cagtttagag cattacaaag gtatgcttta gacgaacata cgctattttg aaaacctaaa 420 ctaacaaata aatatacaaa ttatgaaaat tattctggaa ttcctttaaa attattattt 480 tgaaaagata ttattaacta cccagaatct ttttgatacg ttgtaagtcg tattaataaa 540 gtacgtagaa aaaaagtata ttttactaaa tgttctaaca gaaccttaac tacagctggt 600 tgaactttta ttacaccatt tgcatcaaat gtaaaatata caggtattgg tgctcaagat 660 ttactattag tatcagttgt ttttgctggt attagttcta cagtatcgtt tacaaattta 720 ttaattacaa gacgtacttt atgtatgcct ggtatgagac atagaagagt actattacca 780 tttgtaacta taggtatttt ttttgcatta agaatgttgg ctattattac acctgtgtta 840 ggagcggcta tgattatgat ggctttagat agacattgac aaactacatt ttttgatttt 900                                                                          900 <210> 8 <211> 678 <212> DNA <213> Paralichthys olivaceus <400> 8 atgtgtaagg tgctgggggg ccttttcatc gtggcccttt gtttaggagg cgtcctggcg 60 ttttcttggt acacgaacaa atctgagatg atgacgcaat caggccaaac agcggccctg 120 agccagaagg actgcgctga gaaaacagaa ccccacaaca cactgaggca aatcagcagc 180 agagccaagg cggccatcca tttagaaggt agagacgagg aagacgagga gacttccgaa 240 aacaagctgg tgtggaagaa cgacgaaggc ctagcattca ctcagggcgg cttcgaactg 300 gtggacaacc acatcatcat cccacgaagc ggcctctact tcgtctacag ccaggcgtcg 360 ttcagagtct cctgcagcag cgacgatgcc gacgatggca aggaggcagc ggaaaaacac 420 ctcacgtcca tcagccacag ggtatggctc ttcacggagt ccctgggcac ccaagtgtcc 480 ctgatgagcg cggtgaggtc ggcgtgccag aagagtcagg aggacgccta cagagacgga 540 cagggctggt acaatgccat ctacctgggc gcagtgttcc agctgaacga aggggacaag 600 ctgtggacgg agaccaacat gctctcggag ctggagacag agagcggcaa gactttcttc 660 ggtgtctttg cactttga 678 <210> 9 <211> 783 <212> DNA <213> Paralichthys olivaceus <400> 9 atgctcctgt tcatggtggt ggtgatgctg tgcacggagg tcacggtgaa gacctgggct 60 cagtccctgt gtgacccact gactcagtat gaggaggccg gtcagtgcag caagatgtgt 120 ggtccaggca ccaggaggat gagtcaaagc acttgtacgg atcctcagtg cgcagagtgt 180 gggaaccgtg aatatcagga taggtacacc agggaagccc agtgtaaacg tcagccatac 240 tgtgacccga ataaaaacct gcgtgtgacc aagccagaga gcaagacgaa gcaaagcccc 300 tgcatctgcc tgctgggttt ccactgctcc agtggcacgt gtgtcacctg tgtgccgcac 360 accagccact cctggagcag cgtgtgcacc aagtggacgg aatgtgagag tggataccac 420 atccaagaga gtggaacaaa cgaatctgac aacatatgcg tggaacctcc gcggcaccat 480 ggaggtctga ttgcatgtgt tgttgcagtt ggcagtttgg ctgtagttgg actcatggtt 540 tgtctctgca aaggagaaac aaaacaaagg gcaaaggatt atctggaatc gtgccatgga 600 gacaaggaga acctacagcg agagcccagc ctagtgttgt tcaccacgct cgatgagacc 660 gaaaaccatg aactgctgcc gttcacagag gaggaagaga tgaagatacc ggagaaaacc 720 acgaggacca aagggaggag cagttggtgg atgctgttct cagtgacaat ggacagtgtg 780 tga 783 <210> 10 <211> 654 <212> DNA <213> Paralichthys olivaceus <400> 10 atggaatcca agatggaatg caacgtgagc cagatgtgga gcgccaagat gccacaggga 60 ctgaacttgg agatctccca tcacccgatg acaatgagaa gtgtggtcaa cctcatcatc 120 gccatggagc ggctgaaggg cagccattcg gaatctgtgc tgagcacaag cttcacagat 180 aggggcgtgt acacgtgcaa catcactgac agccagaaga ggaacttcat tctggtccag 240 aacagcatgg agctccacgc cgtgatgctg cagggaggca gcagcaaccg caaagttctt 300 ctcaacatgt ccacctatgt gcacccttca cccaccatcg aagccaggcc tgtcgttctg 360 ggcatcaaag acacagactt cttcctgtca tgccagaaga atggtgcaga gccaaccctg 420 catctggagc gtgtcgagaa caaatgcgac ctggaggcat tcagcaggga cagtgagatg 480 gtgcgatttc tgttctacaa gcaggacagc gggggggtga gcatcagcac cctcatgtcg 540 gcccgcttcc ccaactggta catcagcaca tcagagcaag acaacaggcc agtgatggtg 600 ggtcagaaga atgcccggtg ctaccagacc ttcaacatcc agcatcagag ttaa 654 <210> 11 <211> 633 <212> DNA <213> Paralichthys olivaceus <400> 11 atggactctg agacagttgt agctctgctc tactacttcc tgtttgcagt tttggttcaa 60 tcagttccca tcagcccagc gcccaacact cccccggtgt tgaaggaggc agccgagcga 120 gcaaagacgc tggtggagaa aatcctcaga gagctccctg ccgtgcacac tgccaccgtc 180 aacacagiagg gtttgaccct cgaccccgcg cctcagactc caaacctgca gatgatggtg 240 acctccctgg gcatccccgc cacccccatc ctcaaaccac tgtctgaacg cttcacaatg 300 gacatgtgtg tgagtcgtat gtcagtgggc tgtctgttgt accaggggct gctgggagtt 360 ttagctgaca ggctgagtgg actaacgaac ctgcgagctg acctcagaga cttgctgacc 420 cacatcaaca agatgaagga ggcagctcag ttcggcgccg agagtccgga tcagaaccag 480 agtctggatc tggcctctcg tctccatggt aactacgagg tgcaggtggc agtccatgtg 540 acgctgacac agcttcgttc gttctgtcat gacctgatcc gcagtctgag ggccatcgca 600 acctacaggc gccgagctgc aggtgcacgc taa 633 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for Quantitative real-time PCR of the IL-1beta of          Paralichthys olivaceus <400> 12 tccaccagat cagttcagca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for Quantitative real-time PCR of the IL-1beta of          Paralichthys olivaceus <400> 13 gagaagaact ttgcggttgc 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the IL-6          Paralichthys olivaceus <400> 14 cttcagcaag gaggcttgtc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the IL-6          Paralichthys olivaceus <400> 15 ctttgatgag gccgatcagt 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the TLR2          Paralichthys olivaceus <400> 16 ctccacaaac tgacccacct 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the TLR2          Paralichthys olivaceus <400> 17 ctccagagtt ttgggcagac 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the TLR3 of          Paralichthys olivaceus <400> 18 accgacgagc tgtctcctta 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the TLR3 of          Paralichthys olivaceus <400> 19 ctctcgtctg cttggtcctc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the TNF          Paralichthys olivaceus <400> 20 cacatcatca tcccacagag 20 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the TNF          Paralichthys olivaceus <400> 21 cgtgaagagc catacctgt 19 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the GCSF          Paralichthys olivaceus <400> 22 gcagttaccg gctaatccaa 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the GCSF          Paralichthys olivaceus <400> 23 tagcatgtgg cctagctcct 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the NKEF          Paralichthys olivaceus <400> 24 ccactggact tcaccttcgt 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the NKEF          Paralichthys olivaceus <400> 25 ccactggact tcaccttcgt 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the beta-actin of          Paralichthys olivaceus <400> 26 tttccctcca ttgttggtcg 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> a primer for quantitative real-time PCR of the beta-actin of          Paralichthys olivaceus <400> 27 gcgactctca gctcgttgta 20 <210> 28 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, CD40 <400> 28 ggcgggcgga attcatgctc ctgttcatgg tggtg 35 <210> 29 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, CD40 <400> 29 ggcgggcgga attccacact gtccattgtc actga 35 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, TNF <400> 30 ggcgggcgga attcatgtgt aaggtgctgg ggggc 35 <210> 31 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, TNF <400> 31 ggcgggcgct cgagaagtgc aaagacaccg aaga 34 <210> 32 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, IL-1beta <400> 32 ggcgggcgga attcatggaa tccaagatgg aatgc 35 <210> 33 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, IL-1beta <400> 33 ggcgggcgct cgagactctg atgctggatg ttgaa 35 <210> 34 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, GCSF <400> 34 ggcgggcgga attcgacgac gacgacaaga tggactctga gacagt 46 <210> 35 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> a primer of Construction of the Recombinant protein, GCSF <400> 35 ggcgggcgct cgaggcgtgc aggtgcagct cggcg 35

Claims (11)

Culturing a sutucca insect comprising the nucleotide sequence of SEQ ID NO: 6 to obtain a culture;
Adding and reacting formalin to the culture; and
And obtaining a scuticacid inactivated liquid from the formalin-added culture
A method for producing a vaccine of aquaculture for a scutica.
The method according to claim 1,
Wherein the aquaculture fish is selected from the group consisting of flounder, perch, dome, rockfish, basset, and blowfish.
The method according to claim 1,
Wherein said Sukitika spp. Is Miamiensis avidus. &Lt; Desc / Clms Page number 19 &gt;
The method according to claim 1,
Wherein the formalin is added in an amount of 0.1 to 0.8% by weight based on the cultured product.
The method according to claim 1,
A method for producing a vaccine for aquaculture fish, comprising adding formalin to the culture and reacting for 5 to 20 hours.
The method according to claim 1,
Further comprising the step of centrifuging the obtained scuticaca inactivated liquid to obtain a precipitate.
The method according to claim 1,
Further comprising the step of centrifuging the scuticacid inactivating solution to obtain a precipitate and preparing a vaccine using the obtained precipitate.

The method according to claim 1,
Wherein the vaccine is administered to the fish through injection into the fish.
The method according to claim 1,
Wherein the culturing is performed for 7 days to 80 days.
The method according to claim 1,
Wherein the vaccine comprises or does not comprise an adjuvant.


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