KR101486236B1 - Vaccine adjuvants comprising novel peptide from rock bream - Google Patents

Abstract

The present invention relates to a vaccine adjuvant for fish, which contains, as an active ingredient, a vaccine adjuvant for fish, which contains an effective ingredient of a protein derived from Oplegnathus fasciatus which is capable of effectively promoting the immunity of fish, The method comprising the steps of:

Description

{Vaccine adjuvants containing novel peptide from rock bream}

TECHNICAL FIELD The present invention relates to a vaccine adjuvant for fish, and more particularly, to a vaccine adjuvant for fish containing a protein derived from a dolomite as an active ingredient.

The Korean aquaculture industry has developed steadily with the development of science and technology. However, the productivity of the aquaculture industry has been decreasing due to various infectious diseases in recent years. Representative bacterial diseases of fish include Edwasdsiellosis , Streptococcosis , and Vibriosis . In order to prevent these diseases, various inactivated vaccines have been developed in Korea and are being used at the aquaculture site. However, they are putting a lot of economic burdens on the fishermen due to the additional vaccination to increase the vaccine efficacy and duration. Vaccines have limitations in preventing fish diseases that are more prone to multiple infections than simple infections, because they only augment specific immune responses against a single disease for which the pathogen is known. For this reason, there has been a constant demand for improvement of the vaccine effect. As an alternative to such a demand, a variety of physiologically active substances are used to activate the immune response of the fish, thereby improving the resistance against multiple fish disease infections (Adjuvant ) Has become a necessity for development.

Adjuvants such as aluminum hydroxide gel or mineral oil emulsion, which have been used as vaccine adjuvants to date, have been reported to increase cell-mediated immune responses as well as humoral immunity. However, several side effects And it is required to develop new adjuvant to increase the effect of the vaccine. In mammals, the development of recombinant proteins from various immune-related genes for the development of new immunoadjuvants has been studied as an adjuvant. However, the study of molecular adjuvants for enhancing the efficacy of vaccines in fish It is an early stage.

Since the vaccine enhances a specific immune response only for a single disease, it has limitations in preventing fish diseases where complex infection is more likely to occur than simple infection. For this reason, improvement of the vaccine effect is required. As an alternative, there is a demand for the development of a vaccine adjuvant for fish which can enhance the resistance to multiple fish disease infections by activating the immune response of the fish using various physiologically active substances.

The present inventors first identified immune-stimulating effects of CC chemokine 1 protein and TRx (Thioredoxin) 1 protein, which are proteins derived from the sea tangle, as a fish vaccine capable of activating the immune response of the fish and enhancing resistance to multiple fish disease infections It is intended to provide an adjuvant. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a vaccine adjuvant for fish, which comprises a protein derived from Olegnathus fasciatus as an active ingredient.

The calf-derived protein may be a CC chemokine 1 protein or a TRx (Thioredoxin) 1 protein.

The CC chemokine 1 protein may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 1, and the TRx (Thioredoxin) 1 protein may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 2. The inventors of the present invention found that when the CC chemokine 1 protein or TRx (Thioredoxin) 1 protein is used together with a vaccine for fish, the immune response of the fish is activated as the effect of the vaccine continues, Resistance to infections, and can reduce the mortality of fishes for the first time. The infectious disease may include, but is not limited to, Edwasdsiellosis, Streptococcosis, Vibriosis, and Slide Bacillus.

In the vaccine adjuvant for fish, the effective dose of the protein derived from Dolomite according to an embodiment of the present invention may be 0.001 mg / ml to 1 mg / ml, more preferably 0.05 mg / ml to 0.5 mg / ml Lt; / RTI > On the other hand, the dose can be appropriately adjusted according to the state of the fish.

In the vaccine adjuvant for fish, the protein derived from Dolomite according to an embodiment of the present invention can be administered orally or parenterally, and can be administered by intraperitoneal injection at the time of parenteral administration, or may be added to the feed.

According to one aspect of the present invention, there is provided a method for immunizing a fish, comprising the step of administering a vaccine adjuvant for fish, together with a fish protein derived from the sea tangle, to the fish together with the fish vaccine composition.

According to one embodiment of the present invention as described above, it is possible to implement an immunosuppressant that can economically and effectively enhance the immunity of a fish. Of course, the scope of the present invention is not limited by these effects.

FIG. 1 is a graph showing an increase in the efficiency of a vaccine by recombinant CC chemokine 1 protein (rRbCC1) according to an embodiment of the present invention through agglutination reaction.
FIG. 2 is a graph showing an increase in the efficiency of the vaccine by the recombinant TRx1 protein (rRbTRx1) according to an embodiment of the present invention through agglutination.
FIG. 3 is a graph showing the results of artificial infection of S. iniae FP5228 with a recombinant CC chemokine 1 protein (rRbCC1) in combination with a vaccine ( S. iniae FKC) according to an embodiment of the present invention, Fig.
FIG. 4 is a graph showing the results of a mortality rate of S. iniae FP5228 after transfection with a recombinant TRx1 protein (rRbTRx1) and a vaccine ( S. iniae FKC) FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It should be understood, however, that the invention is not limited to the disclosed embodiments and examples, 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, It is provided to fully inform the owner of the scope of the invention.

Example 1: Synthesis of Protein Derived from Dolomite (rRbCC1, rRTRx1)

CC chemokine 1 cDNA derived from Rock bream has been identified by EST (expressed sequence tag) analysis of a dodomain cDNA library stimulated with LPS (lipopolysaccharide, lipopolysaccharide) in a previous study of the present inventor (Kim JW , et al., J. Fish pathol., 23: 229-38, 2010). The CC chemokine 1 polypeptide derived from the dorsal root was referred to the present invention as SEQ ID NO: 1, and the TRx (Thioredoxin) 1 polypeptide was referred to the present invention as SEQ ID NO: 2.

Example 2: Breeding of dolphins

Dodom (about 70 g) was received from Gyeongsangnam-do Fishery Resources Research Institute of Tongyeong. The red sea bream was raised in a 5-ton water tank supplied with filtered seawater, constantly ventilated during breeding, and supplied commercial food commercially available three times a day. Sea water was maintained at 23 ± 1 ℃. In the following Experimental Example, the vaccination was performed for about 2 weeks before the vaccination.

EXPERIMENTAL EXAMPLE 1: Analysis of the Effect of the Dolomotid Derived Protein as a Vaccine Adjuvant by the Agglutination Reaction

In order to measure the efficiency of rRbCC1 protein or rRbTRx1 protein as a vaccine adjuvant according to one embodiment of the present invention, a vaccine against streptococcosis, which is a major cause of death of Korean farmed fish species, Lt; / RTI > The rRbCC1 protein was administered in combination with the vaccine to the dams, followed by serum agglutination experiments.

First, the strain S. iniae FP5228 was purchased from the Department of Pathology, National Fisheries Research and Development Institute to prepare a formalin-killed cell (FKC) to be used as a vaccine. The cultured bacteria were suspended in 1.5% (w / v) NaCl (BHIB) liquid medium (Difco, USA). The bacteria that had been mass-cultured were collected by centrifugation, suspended in phosphate buffered saline (PBS), formalin added to a final concentration of 4%, inactivated at room temperature for 48 hours Then, it was washed three times with PBS, and resuspended in PBS to prepare S. iniae FKC.

Thereafter, serum cohesion was observed by the combination of S. iniae FKC alone, S. iniae FKC and rRbCC1 protein, or S. iniae FKC and rRbTRx1 protein. The S. iniae FKC single dose group was administered once through the abdominal cavity at a time of 100 μl (1 mg of vaccine per fish) simultaneously with the start of the experiment. In the group administered with the S. iniae FKC and rRbCC1 protein, 100 μl FKC and rRbCC1 protein (100 μg / ml) were intraperitoneally administered once, and 100 μL FKC and rRbTRx1 protein (10 μg / ml) were administered intraperitoneally to the peritoneal cavity together with the S. iniae FKC and rRbTRx1 protein combination group And immunization test was performed for 4 weeks.

Nine animals were collected every week for each experimental group, and blood samples were taken from the subarachnoid veins and left at 4 ° C for 4 hours. The serum was then separated by centrifugation and transferred to a new flash tube.

After that, serum aggregation concentration of S. iniae was analyzed by microtiter assay (Cho MY, et al., J. Fish pathol. , 19 (2): 165-72 , 2006) 50 μl of each of the sera obtained through centrifugation was sequentially diluted in 50 μl of PBS, and 50 μl of S. iniae FP5228 FKC (OD 0.8 at 600 nm) was added thereto. Plates were left overnight at room temperature and observed for aggregation reaction through a microscope. Antibody titer was expressed as the reciprocal of the highest dilution factor of the indicated serum.

As a result, as shown in Fig. 1, when the vaccine was administered and changes were observed for 4 weeks, serum aggregation between the single administration of FKC and the combination administration of FKC and the recombinant protein rRbCC1 or rRbTRx1 protein was not statistically significant However, when the FKC vaccine and rRbCC1 protein were administered in combination with the FKC single vaccine administration, it was observed that serum aggregation was maintained for a longer period of time (see FIGS. 1 and 2). These results suggest that the rRbCC1 and rRbTRxl recombinant proteins according to one embodiment of the present invention may function as an adjuvant to maintain the effect of the vaccine.

Experimental Example 2: Toxicity analysis of fish protein derived from dolomite through hematological index analysis

The hematological indices of rdbCC1 or rRbTRx1 protein according to one embodiment of the present invention were examined to determine the toxicity of the protein derived from the dolomite.

First, the recombinant protein rRbCC1 was prepared at a high concentration of 200 μg / ml (2-fold), 500 μg / ml (5-fold), and 1,000 μg / ml (10-fold) . The recombinant protein rRbTRx1 was prepared at a high concentration of 20 μg / ml (2-fold), 50 μg / ml (5-fold), and 100 μg / ml (10-fold) . As a control group, the same amount of PBS was administered. After 24 hours and 48 hours after the administration, blood samples were taken from the subcutaneous vein in each group (n = 3) and allowed to stand at 4 ° C for 4 hours to allow the blood clots to coagulate.

After centrifugation, the serum was separated and transferred to a new tube. Total hematopoietic protein, albumin, alkaline phosphatase, blood urea nitrogen , BUN), lactate dehydrogenase, triglyceride, total cholesterol, aspartate aminotransferase, alanine aminotransferase, and glucose. (Jung SH, et al., J. Fish pathol. , 19 (3): 253-65, 2006). The above indices were analyzed based on the baseline range of an animal dry chemical system of FUJI DRY-CHEM 4000i instrument (FUJI PHOTO FILM Co., Japan)

As a result, as shown in Table 1, when the recombinant protein rRbCC1 was administered for 48 hours, the 200, 500, and 1,000 μg / ml administration groups showed a statistically significant difference between the control group and the hematological index It was not. In particular, there was little change in GPT compared with the PBS-treated control group. In addition, there was no statistically significant difference between the control group and the hematological index in the 20, 50 and 100 μg / ml administration group after 48 hours of administration of the recombinant protein rRbTRx1. In particular, there was little change in GPT compared with the PBS-treated control group. These results indicate that rRbCC1 and rRbTRx1, which are proteins derived from dolomite according to an embodiment of the present invention, are not toxic and can be usefully applied as a vaccine adjuvant.

However, there was a statistically significant difference in the TP values, which was due to the fact that the control values were much lower than normal (see Table 1).

ALB
(g / dL) BUN
(mg / dl) GPT
(U / L) GOT
(U / L) GLU
(mg / dl) TP
(g / dL) TG (mg / dL)
Reference normal range in the veterinary 3.8-5.0
8.0-23
4.0-44
3.0-38
70-110
6.7-8.3
50-149
PBS Control 1.7 ± 0.3 8.3 ± 1.2 11.7 ± 1.2 38.0 ± 6.2 61.3 ± 9.8 3.2 ± 0.3 250.7 ± 27.4 rRbCC1 24h 2-fold 2.3 ± 0.4 11.1 ± 0.8 9.7 ± 0.6 43.3 ± 5.0 73.7 ± 4.5 6.7 ± 0.4 * 314.3 ± 19.1 5-fold 1.7 ± 0.1 8.4 ± 0.3 7.3 ± 1.5 20.7 ± 1.5 45.7 ± 4.2
5.8 ± 0.3 *
403.0 + - 22.1 * 10-fold 3.0 ± 0.6 *
7.9 ± 0.2
19.0 ± 1.0
53.3 ± 18.9 48.0 ± 3.6
6.4 ± 0.4 *
276.3 ± 11.5
rRbCC1 48h 2-fold 2.4 ± 0.4
9.4 ± 1.0
19.0 + 13.5
22.7 ± 4.7 62.7 ± 33.2 7.6 ± 0.7 *
349.3 ± 33.5 * 5-fold 2.1 ± 0.2
9.2 ± 2.1
12.7 ± 4.6
41.3 ± 5.0 31.7 ± 9.1 * 6.4 ± 0.2 *
259.7 ± 33.5
10-fold 2.3 ± 0.2 10.3 ± 5.9 10.0 ± 1.0 33.7 ± 8.4 31.3 ± 1.5 * 6.2 ± 0.4 *
254.7 ± 34.4
rRbTRx1 24h 2-fold 2.0 ± 0.7 8.4 ± 1.1 7.3 ± 1.5 26.0 ± 3.0 40.0 ± 6.2 5.5 ± 0.8 * 346.7 ± 9.5 * 5-fold 2.2 ± 0.3 7.4 ± 0.6 9.0 ± 1.0 31.0 ± 1.0 50.7 ± 6.7 6.4 ± 0.9 * 258.7 ± 15.5 10-fold 2.4 ± 0.7 9.4 ± 0.6 9.3 ± 0.6 43.3 ± 5.7 46.3 ± 4.7 5.7 ± 0.4 * 278.3 + - 6.4 rRbTRx1 48h 2-fold 2.4 ± 0.6 10.8 ± 2.2
15.0 ± 8.5
45.3 ± 29.7 34.7 ± 18.6 * 7.6 ± 0.9 *
296.0 ± 80.3
5-fold 2.3 ± 0.3 11.7 ± 0.8
10.7 ± 8.1
26.6 ± 10.5 38.7 ± 9.9
5.9 ± 1.4 *
237.7 ± 83.1
10-fold 2.5 ± 0.6 * 14.7 ± 4.5 *
16.3 ± 9.5
47.717.2
42.0 ± 32.9 6.5 ± 0.8 *
215.0 ± 50.9

Experimental Example 3: Analysis of the reduction effect of mortality due to administration of rRbCC1 and vaccine

In order to analyze the effect of the recombinant protein rRbCC1 or rRbTRx1 according to one embodiment of the present invention as a vaccine adjuvant, S. iniae was immunized against vaccine alone, rRbTRx1 combination, or rRbTRx1 combined immunization for 4 weeks. Survival rates of dorado were observed in each experimental group after artificial infection.

Specifically, the mice were treated with 100 μl of S. iniae FKC vaccine (1 mg / mouse) + rRbCC1 (100 μg / ml) or S. iniae FKC vaccine (1 mg / mouse) + rRbTRx1 (10 μg / ml) For comparison, 100 μl of S. iniae FKC vaccine (1 mg / 1 mouse) was administered alone. As a control, 100 μl of PBS was intraperitoneally administered for 1 week, and the live S. iniae FP5228 strain was treated with 2 X 10 < ⁷ > cells / ml (OD value at 600 nm 1.0).

The water tanks were grouped for each experimental group, and the animals were kept in an 80L FRP tank for 10 days. Symptoms, disease progression and mortality were recorded daily, cumulative mortality was recorded, and relative percent survival (RPS) 1. This experiment was repeated three times.

As a result, as shown in FIG. 3 and FIG. 4, the PBS-injected control group showed a survival rate of 5.6 ± 3.8%, and FKC alone showed 70 ± 3.3%, and FKC and rRbCC1 The survival rate was 87.8 ± 5.1% in the mixed group and 86.7 ± 5.8 in the mixed group of FKC and rRbTRx1. Also, the relative survival rate (RPS) derived from the formula (1) was 68.2% in the FKC monotherapy group, but 87% in the FKC and rRbCC1 group, and FKC and rRbTRx1 And 85.9% in the co-administered group, respectively. As shown in Table 2, RPS was significantly increased when coadministered with the protein derived from the dolomite. 1, 2 and 3 in the following Table 2 indicate the number of repeated experiments, and *, which means statistical significance, means P <0.05 and ** means P <0.01.

These results indicate that rRbCC1 and rRbTRx1 recombinant proteins according to the present invention can be usefully applied as vaccine adjuvant for enhancing the effect of vaccine, and can be applied to fish pathogenic microorganisms such as S. iniae It can provide an effect that stimulates the innate immunity and the nonspecific immune system.

Experimental group n Number of surviving fishes Survival rate (%) Average survival (%) RPS
valence
(Control / FKC) Control-1 30 3 10
5.6 ± 3.8 Control-2 30 One 3.3 Control-3 30 One 3.3 FKC-1 30 20 66.7
70 ± 3.3

68.2

**
FKC-2 30 22 73.3 FKC-3 30 21 70 FKC + rRbCC1-1 30 28 93.3
87.8 ± 5.1
87

** / *
FKC + rRbCC1-2 30 25 83.3 FKC + rRbCC1-3 30 26 86.6 FKC + rRbTRx1-1 30 28 93.3
86.7 ± 5.8

85.9

** / *
FKC + rRbTRx1-2 30 25 83.3 FKC + rRbTRx1-3 30 25 83.3

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the invention. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Vaccine adjuvants comprising novel peptide from rock bream <130> PD13-0658 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 122 <212> PRT <213> Oplegnathus fasciatus <400> 1 Met Val Asn Cys Gly Ser Leu Leu Lys Ser Ala Leu Val Ile Leu Ala   1 5 10 15 Leu Val Ala Val Val Gln Pro Gly Ser Ala Pro Glu Lys Leu Ala Ser              20 25 30 Cys Cys Lys Thr Val Ser Asn Gln Lys Ile Thr Glu Pro Ile Leu Gly          35 40 45 Tyr Leu Val Gln Thr Ala Thr Pro Ser Cys Val Arg Ala Val Ile Phe      50 55 60 Gln Thr Glu Ala Gly Leu Phe Cys Ser Gln Val Thr Ala Pro Trp Val  65 70 75 80 Arg Arg Lys Ile Val Ala Phe Glu Arg Ala Lys Ala Leu Ala Ile Pro                  85 90 95 Ser Ser Val Ser Ser Ser Val Le Ser Ser Ser Val Ser Ser Val             100 105 110 Ser Thr Ala Ser Ser Ser Ser Ser Ser         115 120 <210> 2 <211> 108 <212> PRT <213> Oplegnathus fasciatus <400> 2 Met Val Arg Glu Val Glu Asn Leu Asp Glu Phe Lys Ala Ile Leu Lys   1 5 10 15 Glu Ala Gly Asp Arg Leu Val Val Val Asp Phe Thr Ala Thr Trp Cys              20 25 30 Gly Pro Cys Lys Met Ile Gly Pro Glu Phe Glu Arg Leu Ser Lys Leu          35 40 45 Pro Glu Asn Lys Asn Val Ile Phe Leu Lys Val Asp Val Asp Glu Ala      50 55 60 Ser Asp Val Ser Glu His Cys Lys Ile Ser Cys Met Pro Thr Phe His  65 70 75 80 Phe Tyr Lys Asn Glu Ala Lys Val Ser Glu Phe Ser Gly Ala Asn Lys                  85 90 95 Asp Thr Leu Ala Glu Lys Val Glu Ala Leu Arg Thr             100 105

Claims (5)

A vaccine adjuvant for fish comprising, as an active ingredient, TRx (Thioredoxin) 1 protein derived from Oplegnathus fasciatus having an amino acid sequence represented by SEQ ID NO: 2. delete delete delete A method for immunizing a fish, comprising the step of administering the vaccine adjuvant for fish according to claim 1 to a fish vaccine composition and a fish.

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