Fishing vaccine composite adjuvant, corresponding application and turbot aeromonas salmonicida vaccine

Abstract

The invention relates to a fishing vaccine composite adjuvant. The fishing vaccine composite adjuvant comprises inulin and vegetable oil. The invention provides an application of the fishing vaccine composite adjuvant in fish vaccines. The invention further provides a corresponding turbot aeromonas salmonicida vaccine. According to the fishing vaccine composite adjuvant and the corresponding application thereof, the turbot aeromonas salmonicida vaccine has the advantages that the safety of the vaccine is improved, and the residue of harmful substances in mineral oil is overcome; the stimulation of the vaccine on the body is reduced, and the side effect of the vaccine is reduced; the innate immunity of the scophthalmus maximus is more strongly stimulated, and a stronger immune protection effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a composite adjuvant for fishery vaccines and corresponding applications, and a turbot salmonicidal Aeromonas vaccine, wherein the composite adjuvant comprises inulin and vegetable oil. Background Art

[0002] Turbot (Scophthalmus maximus) belongs to the class of bony fishes, order of Pleurotiformes, family of Flatfish, genus of Turbot. It is native to the Atlantic Ocean in Europe and is known as "turbot" in China. Due to its fast growth rate, strong resistance to stress, and delicious meat, the scale of its farming has been continuously expanded, making it an important marine economic farmed fish in my country. With the scale and intensification of aquaculture, as well as the high density of farming, farmed fish are extremely vulnerable to diseases. During the farming process, turbot is plagued by a variety of pathogens, including Aeromonas, Vibrio, Edwardsiella, etc. Among them, Aeromonas salmonicida is the main pathogen of turbot scabies.

[0003] Inactivated vaccines refer to vaccines obtained by inactivating microorganisms through physical or chemical methods. Inactivated vaccines are highly safe and simple to manufacture, and are the main choice for fish vaccine production. However, inactivated vaccines mainly produce humoral immunity-based immune responses. Aeromonas salmonicida is an intracellular and extracellular pathogen, and simple humoral immunity cannot fully prevent and treat Aeromonas salmonicida. In order to assist inactivating inactivated vaccines to activate cellular immunity, adjuvants are usually the choice.

[0004] Adjuvants, or non-specific immunoproliferative agents, refer to auxiliary substances that are injected into the body together with antigens or in advance to enhance the body's immune response to antigens or change the type of immune response. Currently, most vaccines or adjuvants are developed for humans or livestock. Compared with mammals or birds, the immune system of fish is more inferior. In addition, there are many types of fish and there are differences between species. Therefore, adjuvants developed for mammals are not necessarily suitable for fish. It is very necessary to develop vaccines or adjuvants suitable for fish.

[0005] At present, the adjuvants used in commercial fishery vaccines internationally are mostly mineral oil adjuvants. The body temperature of fish is lower than that of mammals, and the metabolic level is also lower. Mineral oil adjuvants are easy to remain in the body, and the aromatic compounds in them are difficult to metabolize, posing a potential risk to food safety. At the same time, the long-term retention of mineral oil at the injection site can easily cause tissue adhesion at the injection site, affecting the growth and development of the host and affecting economic benefits.

[0006] Inulin is a natural polysaccharide, mainly found in Jerusalem artichoke, chicory and other Asteraceae plants, and also in other plants, bacteria and fungi. Inulin has good biocompatibility, is non-toxic, and has biological health functions such as controlling blood lipids. At the same time, in the field of vaccine research, inulin has the function of an immune adjuvant, which can specifically activate the complement bypass pathway, thereby activating the body's immune system and enhancing the body's humoral and cellular immune responses. Inulin is widely available, low-cost, and has high biosafety and tolerance. It has great potential as an immune adjuvant. However, there have been no reports on the use of inulin as an adjuvant in fish, and it is still unknown whether inulin can activate the immune system of fish.

[0007] In addition, different types of adjuvants act in different ways, and multiple types of composite adjuvants may be able to act on different processes at the same time to achieve a complementary effect. The development of composite adjuvants is an important direction for future adjuvant and vaccine development. Summary of the invention

[0008] In order to solve the above problems in the prior art, the present invention provides a composite adjuvant for fishery vaccines and corresponding applications, and a turbot salmonicidal Aeromonas vaccine.

[0009] In order to achieve the above-mentioned object, the first aspect of the present invention provides a composite adjuvant for fishery vaccine, the main feature of which is that it includes inulin and vegetable oil.

[0010] Preferably, the adjuvant is used for turbot salmonicidal Aeromonas vaccine.

[0011] Preferably, the vegetable oil is soybean oil, rapeseed oil, olive oil, sunflower seed oil, sesame oil, cottonseed oil, rice bran oil, peanut oil, camellia oil or corn oil, preferably soybean oil.

[0012] Preferably, the vegetable oil is contained in the oil phase, and the oil phase includes an emulsifier, and the emulsifier accounts for 1% to 20% of the oil phase. The emulsifier can be selected from one of phospholipid emulsifiers, protein emulsifiers, surfactants, polymer emulsifiers, or a combination thereof. Preferably, PGPR is used as an emulsifier, and the content of the emulsifier in the oil phase is 8%.

[0013] The second aspect of the present invention provides the use of the fishery vaccine composite adjuvant in fish vaccines.

[0014] Preferably, the adjuvant is used for turbot salmonicidal Aeromonas vaccine.

[0015] The third aspect of the present invention provides a turbot salmonicidal Aeromonas vaccine, which mainly comprises an aqueous phase containing an antigen and the adjuvant, wherein the aqueous phase contains inulin in the adjuvant, and the antigen concentration is 1×10 7 cells / mL to 1×109 cell / mL, inulin concentration ranges from 0.1 to 100 mg / mL. The injection dose per fish is 0.1 mL.

[0016] Preferably, the antigen concentration is 5×10 8 cell / mL, and the inulin concentration is 2mg / mL, which is the optimal concentration.

[0017] Preferably, the ratio of the water phase to the oil phase is 1 to 10:1 (v / v). Preferably, the ratio of the oil phase to the water phase is 7:3, and the emulsification time is 5 minutes.

[0018] The fourth aspect of the present invention provides a method for preparing a turbot salmonicidal Aeromonas vaccine, the specific steps of which are as follows:

[0019] Preparation of inactivated bacterial solution: Take the salmonidicide Aeromonas strain and streak it on a tryptone soy broth solid medium (TSA) plate, the culture temperature is 22°C, the culture time is 24h, pick a single colony and culture it in a tryptone soy broth liquid medium (TSB) for 24h, the culture temperature is 22°C, and the bacterial solution is inoculated into fresh TSB at 1% (v / v) and continued to be cultured for 24h. After measuring OD600, add 0.1% (v / v) 37% formalin solution at 22°C and inactivate it for 24h. Centrifuge the inactivated bacterial solution at 6000×g, 4°C for 5min, wash it twice with sterile PBS and resuspend the bacterial solution to ensure that the final vaccine antigen concentration is 1×10 7 ~1×10 9 cell / mL;

[0020] Prepare the oil phase: add polyglycerol ricinoleate (PGPR) as an emulsifier to the vegetable oil, wherein the content of the emulsifier in the oil phase is 1-20% (w / w), and filter (0.22 μm filter membrane) to sterilize to obtain the oil phase;

[0021] Preparation of aqueous phase: Add inulin to the aqueous phase containing the antigen at a solid-liquid ratio of 0.1 to 100 mg / mL.

[0022] Mixing and emulsification: The oil phase and the water phase are mixed and sheared and emulsified for 2 to 10 minutes to obtain a vaccine emulsion, wherein the ratio of the oil phase to the water phase is 1 to 10:1 (v / v).

[0023] The beneficial effects of the fishery vaccine composite adjuvant and the corresponding application, and the turbot salmonicidal Aeromonas vaccine of the present invention are: improving the safety of the vaccine, overcoming the residual harmful substances in the mineral oil; reducing the stimulation of the vaccine to the body, alleviating the side effects of the vaccine; and more strongly stimulating the innate immunity of the turbot, with a stronger immune protection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1The survival curves of each group were shown 28 days after immunization.

[0025] Figure 2 is the level of specific antibodies against Aeromonas salmonicida in turbot serum.

[0026] Figure 3 is the lysozyme level of each group.

[0027] Figure 4 A to 4J show the expression levels of various genes in the samples after immunization. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0030] Example 1

[0031] Vaccine preparation

[0032] Preparation of inactivated bacterial solution: Take the Aeromonas salmonicida strain preserved in the laboratory (registration number of China Microbiological Culture Collection Center: CGMCC No.11375) and streak it on TSA plate, culture temperature is 22℃, culture time is 24h, pick a single colony and culture it in TSB for 24h, culture temperature is 22℃, and the bacterial solution is inoculated into fresh TSB at 1% (v / v) and cultured for 24h. After measuring OD600, add 0.1% (v / v) 37% formalin solution at 22℃ and inactivate it for 24h. Centrifuge the inactivated bacterial solution at 6000×g, 4℃ for 5min, wash twice with sterile PBS and resuspend the bacterial solution (FKC) to ensure that the final vaccine antigen concentration is 5×10 8 cell / mL.

[0033] Preparation of SI vaccine: Polyglycerol ricinoleate (PGPR) was added to soybean oil as an emulsifier, wherein the emulsifier accounted for 8% (w / w) of the oil phase, and the oil phase was obtained by filtration (0.22 μm filter membrane) for sterilization. Inulin (Puxitang, China) was added to the aqueous phase containing the antigen at a solid-liquid ratio of 2 mg / mL to obtain the aqueous phase. The oil phase and the aqueous phase were mixed and emulsified for 5 minutes to obtain the vaccine SI, wherein the ratio of the oil phase to the aqueous phase was 7:3 (v / v).

[0034] Preparation of ISA763A vaccine: Same as the preparation steps of SI vaccine, inactivated bacteria solution and commercial non-mineral oil Montanide TM ISA 763A VG (763A; Seppic, France) was shear emulsified to obtain a stable emulsion with a final concentration of 5 × 10 8 cell / mL of vaccine 763A.

[0035] Preparation of SO vaccine: The same preparation steps as above for SI vaccine were used to prepare the inactivated bacterial solution and soybean oil by shear emulsification to obtain a stable emulsion with a final concentration of 5×10 8 cell / mL of vaccine SO.

[0036] Preparation of IN vaccine: Inulin was added to the inactivated bacterial solution, the final concentration of inulin was 2 mg / mL, and the antigen content was 5×10 8 cell / mL, and obtain vaccine IN.

[0037] Example 2

[0038] Vaccination of turbot

[0039] Five groups of turbot were selected and immunized by intraperitoneal injection of 0.1 mL per fish using a syringe. The five groups of experimental fish were FKC group (injection of resuspended bacterial solution), 763A group (injection of vaccine 763A), SO group (injection of vaccine SO), IN group (injection of vaccine IN), and SI group (injection of vaccine SI). The control group was injected with an equal volume of PBS. There were 45 fish in each group, and they were placed in a water tank equipped with flowing seawater. The water temperature was controlled at 16±1℃ and fed every day. Three fish were taken from each group 7, 14, 21, and 28 days after immunization, and blood was centrifuged from the tail vein to obtain serum. The spleen and head kidney were dissected and stored in RNAStore. The serum and tissue were stored at -80℃ for later use.

[0040] Immune protection rate test

[0041] 28 days after vaccination, each group of fish was treated with 1×10 5 cfu / tail of Aeromonas salmonicida was injected intramuscularly. The cumulative mortality of each group after challenge was recorded every day. The immune protection rate was calculated as follows:

[0042] Relative percent survival rate (RPS): RPS = (1-mortality rate of the immunized group / mortality rate of the control group) × 100%.

[0043] The survival curves of each group are as follows Figure 1 As shown in Table 1, the mortality rate and RPS of each group are shown in Table 1. The protective effect of inulin as an adjuvant in the IN group was slightly different from that in the FKC group, and was much lower than that in the SO, 763A, and SI groups, indicating that the use of inulin alone cannot effectively activate the immune protection of turbot. The soybean oil group (SO) alone had a certain protective effect, but it was lower than that in 763A, and the protective effect of the combination of inulin and soybean oil (SI) was stronger than that in 763A. The combination of the two can provide a stronger protective effect, which may be because soybean oil promotes the slow release of inulin, causing long-lasting and strong immune stimulation.

[0044] Table 1 Mortality rate and immune protection rate of each group

[0045]

[0046] Serum antibody titer test

[0047] The antibody titers against Aeromonas salmonicida in the sera of the 7, 14, 21, and 28 dpv groups isolated in Example 2 were determined by enzyme-linked immunosorbent assay (ELISA). 100 μL of 1.0×10 8 CFU / mL FKC suspension in PBS and incubated overnight at 4°C. The microwells were washed with PBST (PBS containing 0.05% Tween20) and blocked with PBSTA (PBST containing 1% BSA). The turbot serum of each group was diluted in PBST and incubated with 100 μL / well for 3 h at room temperature. The microplate was washed three times with PBST, 100 μL / well mouse anti-turbot IgM (Aquatic Diagnostics Ltd, UK, diluted 1:33 with PBSTA) was added, and incubated for 1 h at room temperature. Subsequently, the microplate was washed again and incubated with 100 μL / well goat anti-mouse IgG conjugated with HRP (Abgent, USA, diluted 1:200 with PBST) for 1 h. Finally, the microplate was washed 5 times, developed with 100 μL / well TMB in the dark for 10 min at room temperature, and the reaction was stopped with 50 μL / well sulfuric acid (2M). The absorbance at 450 nm was read using an enzyme reader.

[0048] The results are as follows Figure 2 As shown, the antibody level in serum increased with the immunization time. At 28 dpv, SO group, 763A group and SI group could induce strong serum antibody level, indicating that the sustained release effect of oil adjuvant can effectively activate humoral immune response.

[0049] Serum lysozyme assay

[0050] The activity of lysozyme was determined by turbidimetry. 10 μL of each group of serum from 7, 14, 21, and 28 dpv separated in Example 2 was placed in the wells of a 96-well plate and stored at low temperature on ice. Resuspend the lysozyme micrococcus with PBS to adjust the absorbance to about 0.4 at 570 nm. Add 190 uL of lysozyme micrococcus solution to each well and immediately read at 570 nm with a microplate reader. Measure once every 3 minutes for 30 minutes. Calculate the slope to represent the activity of lysozyme, and the ratio of the lysozyme activity of each group to PBS is used as the difference multiple.

[0051] Lysozyme activity Figure 3As shown in the figure, at 7 and 14 dpv, the lysozyme level in the IN group increased significantly and then decreased, indicating that inulin can effectively activate the innate immune response in the short term, but the duration is short. At 21 and 28 dpv, the serum lysozyme content in SI was significantly higher than that in other groups. Soybean oil can make the innate immune response induced by inulin last longer and provide stronger protective effects.

[0052] Example 3

[0053] Gene expression level

[0054] According to the manufacturer's instructions, Trizol (Invitrogen, USA) was used to extract total RNA from spleen and head kidney of each group dissected at 7, 14, 21, and 28 dpv in Example 2. Subsequently, the extracted RNA samples were reverse transcribed using FastKing RT Kit (Tiangen, China) according to the manufacturer's instructions. The final cDNA reaction mixture (20 μL) was diluted with 80 μL of water and stored at -20°C until use.

[0055] The Green qPCR SuperMix Kit (TransGen, China) was used to analyze the gene expression level. -ΔΔ The primers used were shown in Table 2 below.

[0056] Table 2 Primers

[0057]

[0058] The specificity of the primers was tested by melting point curve method with β-actin as the control. Figure 4 A to Figure 4 As shown in Figure J, compared with the control group, the expression levels of IL-1β, TNFα, and IFNγ in each group were increased, indicating that the vaccine effectively stimulated the inflammatory response, while the expression levels of complement C3 and complement factor B (CFB) in the SI group were higher than those in other groups, which shows that SI can effectively activate the body's complement response, thereby enhancing the protective effect of the vaccine.

[0059] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. A composite adjuvant for fishery vaccines, characterized in that: Includes inulin and vegetable oils.

2. The fishery vaccine composite adjuvant according to claim 1, characterized in that: The adjuvant is used for turbot salmonicidal Aeromonas vaccine.

3. The fishery vaccine composite adjuvant according to claim 1, characterized in that: The vegetable oil is soybean oil, rapeseed oil, olive oil, sunflower seed oil, sesame oil, cottonseed oil, rice bran oil, peanut oil, camellia oil or corn oil.

4. The fishery vaccine composite adjuvant according to claim 1, characterized in that: The vegetable oil is contained in the oil phase, and the oil phase includes an emulsifier. The content of the emulsifier in the oil phase is 1% to 20%.

5. Use of the fishery vaccine composite adjuvant according to any one of claims 1 to 4 in fish vaccines.

6. The use according to claim 5, characterized in that: The adjuvant is used for turbot salmonicidal Aeromonas vaccine.

7. A turbot salmonicidal Aeromonas vaccine, characterized in that: The invention comprises an aqueous phase containing an antigen and an adjuvant according to any one of claims 1 to 5, wherein the aqueous phase contains inulin in the adjuvant, and the antigen concentration is 1×10 7 cells / mL to 1×10 9 cell / mL, and the inulin concentration ranged from 0.1 to 100 mg / mL.

8. The turbot salmonicidal Aeromonas vaccine according to claim 7, characterized in that: The antigen concentration is 5×10 8 cell / mL, and the inulin concentration was 2 mg / mL.

9. The turbot salmonicidal Aeromonas vaccine according to claim 7, characterized in that: The ratio of the water phase to the oil phase is 1 to 10:1 (v / v).

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