Micropterus salmoides rhabdovirus attenuated freeze-dried vaccine activator and application thereof

Abstract

The invention discloses a micropterus salmoides rhabdovirus attenuated freeze-dried vaccine activator. The micropterus salmoides rhabdovirus attenuated freeze-dried vaccine activator comprises ginsenoside Rg1. The invention further discloses application of the ginsenoside Rg1 in preparation of the micropterus salmoides rhabdovirus attenuated freeze-dried vaccine and a method for activating the micropterus salmoides rhabdovirus attenuated freeze-dried vaccine. After the ginsenoside Rg1 is used for activating the vaccine, the virus titer is high, the high activity of the freeze-dried vaccine can be maintained, the immune protection effect of the largemouth bass rhabdovirus attenuated freeze-dried vaccine can be remarkably improved through soaking immunization, and a foundation is laid for application of the largemouth bass rhabdovirus attenuated vaccine.

Description

Technical Field

[0001] This invention belongs to the field of animal vaccines, specifically relating to an activator for a largemouth bass rhabdovirus attenuated freeze-dried vaccine and its application. Background Technology

[0002] Largemouth bass ( Micropterus salmoides Largemouth bass (Scoidea spp.) is a freshwater aquaculture fish with significant economic value, prized for its delicious flesh and lack of intramuscular bones. According to data from the 2025 *China Fisheries Statistical Yearbook*, the scale of freshwater bass farming in my country has reached over 930,000 tons. However, with the continuous increase in the farming density of largemouth bass in recent years, viral diseases have shown a high incidence trend, among which largemouth bass rhabdovirus (LRV) is particularly prevalent. Micropterus salmoides rhabdovirus Infections caused by MSRV (Medium-total Scale Virus) are particularly prominent. MSRV, due to its high pathogenicity, high mortality rate, and specificity infecting fry, has become one of the major viral diseases restricting the development of the largemouth bass aquaculture industry. This virus primarily infects juvenile fish with a body length of 2-5 cm. Clinical manifestations during infection include significant lethargy, swimming imbalance, abdominal swelling, and body deformities, accompanied by necrotic ulcers and hemorrhagic lesions in multiple organs. The mortality rate after infection is often as high as 80%, causing huge economic losses to the aquaculture industry and becoming a key limiting factor restricting the further development of largemouth bass aquaculture. Currently, there is no effective prevention or treatment method for this disease.

[0003] For the prevention and control of viral diseases in fish, there are two main methods: antiviral drugs and vaccination. Currently, highly effective antiviral drugs are relatively scarce in aquaculture, while vaccination can not only induce long-lasting specific immune protection in fish but also avoid the problem of pathogen resistance that may arise from drug use. Vaccination is one of the effective strategies for preventing and controlling viral diseases. However, MSRV mainly affects the fry stage of bass, and injection immunization is difficult to perform at the fry stage, easily leading to stress and death in young fish, making it difficult to apply to fry. Therefore, immersion immunization is a more feasible immunization method. Among various vaccines, attenuated live vaccines have advantages such as strong immunogenicity, long protection period, and relatively high immune protection rate. In particular, attenuated live vaccines, through immersion immunization, can activate the mucosal immune response of fish through the gills and skin mucosa, thereby achieving simultaneous immunization of fry and the entire fish population. Its ease of operation, batch processing, and low stress significantly improve immunization efficiency.

[0004] To further improve the stability and applicability of vaccines, attenuated live vaccines are often prepared as freeze-dried formulations. However, the reconstitution and activation process of attenuated freeze-dried vaccines can damage viral proteins to some extent, resulting in a decrease in vaccine titer after reconstitution, thus affecting immunogenicity. This leads to a weak immune response induced in fish by immersion immunization and a short duration of activated immune protection. Our laboratory previously successfully isolated an attenuated largemouth bass rhabdovirus strain, MSRV-0509, and confirmed that immersion immunization with this strain can induce effective immune protection. However, the antigen uptake efficiency during immersion immunization is low, resulting in a weak and short-lasting activated immune response. Furthermore, the titer is prone to decrease during the reconstitution of the freeze-dried vaccine, further affecting the immunization effect.

[0005] As a key medium for dissolving and delivering attenuated freeze-dried vaccines for immunization, the role of activators is no longer simply to dilute the vaccine. Suitable activators can not only maintain the vaccine's biological activity but also significantly enhance its immunogenicity. Compared to traditional chemical drugs, traditional Chinese medicine, with its advantages of low toxicity, multi-target effects, bidirectional immunomodulatory properties, and non-addictiveness, is widely used in the field of vaccine adjuvants. Therefore, screening suitable freeze-dried vaccine activators to activate and dissolve attenuated freeze-dried vaccines, maintain vaccine activity, and improve antigen presentation efficiency in fish during immunization is a crucial step in optimizing immersion immunization. Summary of the Invention

[0006] The purpose of this invention is to address the above-mentioned technical problems by providing an activator for a largemouth bass rhabdovirus attenuated freeze-dried vaccine that can effectively maintain vaccine activity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides an activator for a largemouth bass rhabdovirus attenuated freeze-dried vaccine, which includes ginsenoside Rg1.

[0008] Preferably, the activator of the largemouth bass rhabdovirus attenuated freeze-dried vaccine includes an aqueous solution of ginsenoside Rg1 with a concentration of 5-20 mg / mL.

[0009] Secondly, this invention provides the application of ginsenoside Rg1 in the preparation of a largemouth bass rhabdovirus attenuated freeze-dried vaccine.

[0010] Preferably, the working concentration of ginsenoside Rg1 is 5~20 mg / mL.

[0011] Thirdly, the present invention provides a method for activating the largemouth bass rhabdovirus attenuated freeze-dried vaccine, which includes the following steps: dissolving the largemouth bass rhabdovirus attenuated freeze-dried vaccine in an aqueous solution of ginsenoside Rg1.

[0012] Preferably, the largemouth bass rhabdovirus attenuated freeze-dried vaccine has a viral titer of 10 in the viral solution before freeze-drying. 7.3 TCID 50 / mL.

[0013] Preferably, the virus strain in the largemouth bass rhabdovirus attenuated freeze-dried vaccine is largemouth bass rhabdovirus MSRV-0509, which is deposited at the China Center for Type Culture Collection with accession number CCTCC NO: V202412.

[0014] In this invention, activating the vaccine with ginsenoside Rg1 resulted in a high viral titer, maintaining the high viability of the freeze-dried vaccine. Using 20 mg / mL ginsenoside Rg1 to activate the freeze-dried vaccine, immersion immunization achieved optimal immunization efficacy, with a relative immunoprotection rate as high as 97.6%, and significantly upregulated the expression of immune-related factors in tissues such as the spleen, head, kidney, and gills. Therefore, using 20 mg / mL ginsenoside Rg1 as an activator for the freeze-dried vaccine, through immersion immunization, can significantly enhance the immunoprotective effect of the largemouth bass rhabdovirus attenuated freeze-dried vaccine, laying the foundation for the application of largemouth bass rhabdovirus attenuated vaccines. Attached Figure Description

[0015] Figure 1 The following are images of a largemouth bass rhabdovirus attenuated freeze-dried vaccine sample. (A) Sample characteristics before freeze-drying, (B) Sample characteristics after freeze-drying, (C) Sample characteristics after being dissolved in PBS.

[0016] Figure 2 The effects of different activators on CPB cell viability are shown (*). P <0.05、** P <0.01).

[0017] Figure 3 The effect of the activator on the viral titer of the largemouth bass rhabdovirus attenuated freeze-dried vaccine was shown (*). P <0.05、** P <0.01).

[0018] Figure 4 The effects of different types of ginsenosides on the viral titer of a largemouth bass rhabdovirus attenuated freeze-dried vaccine were demonstrated.

[0019] Figure 5 The survival curves of largemouth bass after immunization by soaking in different activators are shown.

[0020] Figure 6The relative expression levels of IFN-γ and IL-12 in the spleen, forekidney, and gills of largemouth bass after immunization with ginsenoside-activated freeze-dried vaccine are shown. (A, D) Expression levels of IFN-γ and IL-12 in spleen tissue; (B, E) Expression levels of IFN-γ and IL-12 in forekidney tissue; (C, F) Expression levels of IFN-γ and IL-12 in gill tissue. Detailed Implementation

[0021] To facilitate understanding of the present invention, a more complete description will be given below with reference to specific embodiments. Preferred embodiments of the invention are shown in the accompanying drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0022] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0023] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0025] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0026] 1. Materials and Methods 1.1 Experimental Materials and Main Reagents Juvenile largemouth bass (1g±0.1g, 3.5cm±0.15cm) were purchased from Guangdong Dequan Fisheries Development Co., Ltd. The Chinese perch brain cell (CPB) line, virulent and attenuated largemouth bass rhabdovirus strain MSRV-0509 were all isolated and preserved in our laboratory. The attenuated rhabdovirus strain MSRV-0509 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO:V202412. In other words, the largemouth bass rhabdovirus was isolated and preserved by the Fish Pathology Laboratory of the Pearl River Fisheries Research Institute. Relevant preservation certificates have been provided in the prior application 202410440997.6.

[0027] CPB cells, or Infectious Splenorenal Necrosis Virus, can be isolated in a laboratory or purchased. This example demonstrates the isolation and preservation in the laboratory of the Fish Disease Laboratory at the Pearl River Fisheries Research Institute, Chinese Academy of Fishery Sciences.

[0028] Main reagents: L15 cell culture medium, trypsin, fetal bovine serum (FBS), and PBS elution buffer were purchased from White Shark Biotechnology Co., Ltd.; ginsenoside Rg1, ginsenoside Rb1, ginsenoside Ro, β-glucan, astragalus polysaccharide, and anisodamine were all commercially available standard products purchased from Shanghai Yuanye Biotechnology Co., Ltd.; RNA extraction reagent was purchased from Chengdu Fuji Biotechnology Co., Ltd.; reverse transcription and RT-qPCR reagents were purchased from TransGen Biotech Co., Ltd.

[0029] 1.2 Preparation of attenuated freeze-dried vaccine for largemouth bass 1.2.1 Cell culture and virus inoculation Monolayers of CPB cells were washed twice with PBS, digested with trypsin, and after complete digestion, L15 medium containing 4% serum was added to adjust the cell concentration to a final concentration of 3.5 × 10⁻⁶ cells / mL. 5 MSRV-0509 was simultaneously inoculated into cells at MOI=0.00001. After the cells were fully infected, the virus solution was stored at -80℃ for later use.

[0030] 1.2.2 Preparation of freeze-dried vaccines The frozen and thawed virus solution was added to a freeze-drying protectant and aliquoted into vials, then incubated at -80°C overnight. The completely frozen virus solution was then placed in a freeze dryer for freeze-drying. After freeze-drying, the vials were sealed and stored at 4°C. The freeze-drying program was set to three stages: -50°C for 2 hours, -40°C for 24 hours, and -25°C for 20 hours.

[0031] The freeze-drying protectants include: 5% trehalose, 10% caseinate, 5% sucrose, 1.5% PEG, and 1% gelatin. These percentages represent the final concentrations of each component in the vaccine semi-finished product. The vaccine semi-finished product refers to the vaccine stock solution before freeze-drying.

[0032] The specific preparation method is as follows: dissolve sucrose and gelatin in water for injection, sterilize by autoclaving to obtain solution A for later use; dissolve trehalose, PEG, and hydrolyzed casein in water for injection, filter and sterilize to obtain solution B for later use; mix solution A, solution B, and virus solution in a volume ratio of 1:1:1 to obtain the vaccine stock solution.

[0033] 1.3 Determination of the working concentration of freeze-dried vaccine activator Cell viability was assessed using the CCK-8 assay. CPB cells were sputtered at a concentration of 3 × 10⁻⁶. 5 Cells were seeded at a concentration of [number] cells / mL in 96-well plates and incubated at 28°C for 12 h. Ginsenoside Rg1, β-glucan, astragalus polysaccharide, and anisodamine were serially diluted 2-fold with water for injection and added to each well in a gradient manner, 100 μL / well, with 6 replicates for each concentration. A blank control group was also included. After 1 h of incubation, the culture medium was discarded, and 100 μL / well of 2% L15 medium was added. The plates were then incubated at 28°C for 24 h. The supernatant was discarded, and the cells were washed three times with PBS. Under light-protected conditions, 100 μL of L15 medium and 10 μL of CCK8 mixture were added to each well. After 4 h of incubation, the absorbance was measured at 450 nm.

[0034] The formula for calculating cell viability is: Cell viability = [(Drug group - Blank control group) / (Cell control group - Blank control group)] × 100%.

[0035] 1.4 Titer determination of freeze-dried vaccine after activation The lyophilized vaccine was dissolved in PBS and aqueous solutions containing ginsenoside Rg1, β-glucan, astragalus polysaccharide, and anisodamine, respectively. CPB cells were seeded into 96-well plates at 100 μL / well and cultured overnight. The culture medium in the 96-well plates was discarded. The lyophilized vaccine was serially diluted 10-fold and added to the 96-well plates at 100 μL / well, with 8 replicates for each concentration. The 96-well plates were placed in an incubator at 28°C and observed continuously for 14 days. The viral titer of each group was calculated using the Reed-Muench method.

[0036] 1.5 Screening of different types of ginsenoside activators The lyophilized vaccines were diluted with PBS and aqueous solutions of three types of ginsenosides Rg1, Rb1, and Ro, respectively. The diluted lyophilized vaccines were then injected into 96-well plates lined with a monolayer of CPB cells at a concentration of 100 μL / well. Each concentration was tested in 8 replicates. The plates were incubated at 28°C for 14 days, and the viral titers of each group were calculated using the Reed-Muench method.

[0037] 1.6 Immersion Immunization Experiment of Fish After Activation of Freeze-Dried Vaccine To determine the immersion immunization effect of different freeze-dried activators on the fish after dissolving the vaccine, largemouth bass were divided into 5 groups of 100 fish per group: PBS control group, PBS-dissolved and activated freeze-dried vaccine group, ginsenoside Rg1 aqueous solution-dissolved and activated freeze-dried vaccine group, β-glucan aqueous solution-dissolved and activated freeze-dried vaccine group, and astragalus polysaccharide aqueous solution-dissolved and activated freeze-dried vaccine group. The vaccine titer in all immunization groups was adjusted to 10. 5 TCID 50 / mL, after the freeze-dried vaccine diluent is fully mixed, each group of largemouth bass is soaked in PBS for 1 hour, and the control group is soaked in PBS for 1 hour. After soaking, each group is transferred to water and soaked for 10 minutes before being placed in a glass tank for rearing. The fish are observed and recorded daily.

[0038] 1.7 Immunoprotective Experiment of Fish Immunized with Freeze-Dried Vaccine On day 21 post-immunization, each group was intraperitoneally injected with a virulent strain of largemouth bass rhabdovirus with an LD50 of 50, at a dose of 50 μL per fish. Morbidity and mortality of the largemouth bass were observed and recorded daily after challenge for two consecutive weeks, and the mortality rate of each group was calculated.

[0039] The relative protection rate (RPS) is calculated using the formula: RPS = [1 - (mortality rate of the immunized group / mortality rate of the control group)] × 100%.

[0040] 1.8 Measurement of immune-related factors In the "1.6 Fish Immersion Immunization Experiment after Activation of Freeze-dried Vaccine" experiment, three largemouth bass from each group were randomly sacrificed at 0h, 4h, 8h, 12h, 24h, and 48h post-immunization. Spleen, head kidney, and gill filaments were collected, labeled, and stored at -80℃. RNA was extracted using the Animal Total RNA Isolation Kit Instruction Manual. The concentration of the extracted RNA was determined using a nucleic acid analyzer, and the RNA from each group was adjusted to a uniform concentration. Finally, cDNA was synthesized using the TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR reverse transcription kit. The expression of IFN-γ, IL-12, and IgM immune genes in the spleen and head kidney was detected by SYBR fluorescent dye qPCR (primer sequences are shown in Table 1).

[0041] The reaction program was: 95℃ for 30s; 95℃ for 5s, 60℃ for 30s, for a total of 40 cycles. The melting curve phases included: 95℃ for 15s, 60℃ for 1min, and 95℃ for 15s.

[0042] The internal reference gene is 18S, using 2 -△△CTThe relative expression levels of genes in each group were calculated using this method.

[0043] Table 1. Primer Information

[0044] 1.9 Data Analysis and Graphing Experimental data were statistically analyzed using GraphPad Prism 10.4.1 software. One-way ANOVA was performed on data with the same indicators in each group, and LSD multiple comparison tests were used to determine the significance of differences. The results are expressed as mean ± standard deviation (mean ± SD). P <0.05 indicates a significant difference. P <0.01 indicates a highly significant difference.

[0045] 2 Results 2.1 Characteristics of freeze-dried vaccines To verify the effect of freeze-drying on vaccine shape, samples before and after freeze-drying were tested for appearance, solubility, remaining moisture, and titer decrease. Results showed that the freeze-dried product maintained good shape, appearing as a white, spongy, loose cake, and dissolved rapidly upon addition of PBS. The remaining moisture content of all freeze-dried samples was below 4%, meeting the requirements for biological products in the Chinese Veterinary Pharmacopoeia. Figure 1 ).

[0046] Based on the virus titer test results, the titers of samples 1 and 2 after lyophilization decreased from 10 to 20, respectively. 8.571 TCID 50 / mL, 10 8.5 TCID 50 / mL decreased to 10 8.219 TCID 50 / mL, 10 8.1 TCID 50 / mL, the viral titer decreased only slightly (Table 2).

[0047] Table 2. Results of virus titer detection before and after freeze-drying of largemouth bass rhabdovirus attenuated freeze-dried vaccine.

[0048] 2.2 Screening of freeze-dried vaccine activators 2.2.1 Determination of the working concentration of the freeze-dried vaccine activator To determine the working concentration of each activator group, cell morphology observation and CCK8 assay were used.

[0049] The results are as follows Figure 2As shown, CPB cells exhibited normal morphology and viability at concentrations of 5–20 mg / mL ginsenoside Rg1 aqueous solution and β-glucan aqueous solution; cells with normal state and viability at concentrations of 5–10 mg / mL astragalus polysaccharide aqueous solution showed significantly decreased viability at a concentration of 20 mg / mL, and significant growth inhibition at 40 mg / mL. P <0.01); 5 mg / mL anisodamine caused a significant decrease in cell viability ( P <0.01), accompanied by cell shrinkage and rounding and agglomeration.

[0050] Therefore, the working concentrations of ginsenoside Rg1 and β-glucan were determined to be 20 mg / mL, and the working concentration of astragalus polysaccharide was determined to be 10 mg / mL.

[0051] 2.2.2 Effect of freeze-dried vaccine activator on vaccine titer To investigate the effect of the activator on the titer of the freeze-dried vaccine, the titer was measured separately.

[0052] The largemouth bass rhabdovirus attenuated freeze-dried vaccine had a viral titer of 10 in the viral fluid before freeze-drying. 7.3 TCID 50 / mL.

[0053] The results are as follows Figure 3 As shown, the titers of the blank control group, the 20 mg / mL ginsenoside Rg1 vaccine group, the 20 mg / mL β-glucan vaccine group, the 10 mg / mL astragalus polysaccharide vaccine group, and the 5 mg / mL anisodamine vaccine group were 10, respectively. 6.289 TCID 50 / mL, 10 6.882 TCID 50 / mL, 10 6.802 TCID 50 / mL, 10 6.59 TCID 50 / mL, 10 6.425 TCID 50 / mL, among which the ginsenoside Rg1 vaccine group and the β-glucan vaccine group had the highest virus titers and the best effect on maintaining the viability of the freeze-dried vaccine.

[0054] 2.2.3 Effects of different types of ginsenoside vaccine activators on vaccine titers To screen for the optimal type of saponin, the effects of three ginsenosides (Rg1, Rb1, and Ro) on the titer of freeze-dried vaccines were compared, and the titers were measured respectively.

[0055] The largemouth bass rhabdovirus attenuated freeze-dried vaccine had a viral titer of 10 in the viral fluid before freeze-drying. 7.85 TCID 50 / mL.

[0056] The results are as follows Figure 4 As shown, the titers of the blank control group, the 20 mg / mL ginsenoside Rg1 group, the 20 mg / mL ginsenoside Rb1 group, and the 20 mg / mL ginsenoside Ro group were 10, ... 7.03 TCID 50 / mL, 10 7.571 TCID 50 / mL, 10 7.429 TCID 50 / mL, 10 7.194 TCID 50 / mL, among which ginsenoside Rg1 has the best effect on preserving vaccine activity after being dissolved in freeze-dried vaccine.

[0057] Therefore, ginsenoside Rg1 was determined to be the optimal activator for freeze-dried vaccines.

[0058] 2.4 Optimized Screening of Freeze-Dried Vaccine Activator Immersion Immunization To verify the immersion immunization effect of the freeze-dried vaccine after activation, the freeze-dried vaccine was dissolved in different freeze-dried vaccine activators and immersed in the vaccine for immunization, followed by challenge on day 21.

[0059] The results are as follows Figure 5 As shown, the relative immunoprotective rates of the PBS-resolved and activated lyophilized vaccine group, the 20 mg / mL ginsenoside Rg1-resolved and activated lyophilized vaccine group, the 20 mg / mL β-glucan-resolved and activated lyophilized vaccine group, and the 10 mg / mL astragalus polysaccharide-resolved and activated lyophilized vaccine group were 88.9%, 97.6%, 92.5%, and 90.8%, respectively. The results confirm that the ginsenoside Rg1-resolved and activated lyophilized vaccine group had the best immunogenicity, with a protection rate as high as 97.6%.

[0060] Therefore, immersion immunization with ginsenoside-activated and dissolved freeze-dried vaccine is the optimal immunization procedure.

[0061] 2.5 Immune Factor Detection The evaluation of vaccine efficacy is not limited to the rate of immune protection; the type and dynamic process of the immune response induced after immunization are also important factors in assessing immunization effectiveness. RT-qPCR detection of immune gene transcription levels is an effective strategy for evaluating vaccine-induced immune responses and the synthesis levels of related proteins.

[0062] To validate the immune response induced by the optimized immunization protocol, immune factors were detected. The effectiveness of the vaccine in activating the immune response was assessed by detecting the expression of cytokines related to the gills and core immune organs (spleen and prokidney) of largemouth bass.

[0063] The results are as follows Figure 6As shown, in the group immunized with 20 mg / mL ginsenoside Rg1-activated lyophilized vaccine, IFN-γ and IL-12 levels in the spleen began to rise at 4 hours after immersion immunization, reaching peak levels at 12 hours, increasing by 20.05 times and 230.64 times, respectively. P <0.01); In the head kidney, IFN-γ was significantly upregulated from 24 h, reaching a peak at 48 h, which was 21.22 times that of the control group ( P <0.01); IL-12 peaked at 24h, 183.66 times that of the control group ( P <0.01 indicates that the antigen was effectively presented to secondary lymphoid organs, activating a strong immune response. In gill tissue, the expression levels of IFN-γ and IL-12 were continuously upregulated after immunization. IFN-γ expression was significantly upregulated at 12 and 24 hours, reaching a peak at 12 hours, which was 12.16 times that of the control group. P <0.01), IL-12 levels were significantly increased at 4, 24, and 48 hours, with a peak at 4 hours, which was 384.87 times that of the control group ( ). P <0.01). Overall, the transcriptional level of immune factors increased significantly from 4 hours onwards, indicating that mucosal immunity was rapidly activated and played a key role in the early defense of the fish.

[0064] In summary, IFN-γ and IL-12 were significantly upregulated in the spleen, head kidney, and gills, with IL-12 showing the most significant upregulation across all tissues (230.64 times higher in the spleen than the control group, 183.66 times higher in the head kidney, and 384.87 times higher in the gills). Given that the immune factors IFN-γ and IL-12 are mainly secreted by Th1 cells and play a crucial role in cellular immune responses, this study confirms that the lyophilized vaccine activated by ginsenoside Rg1 activated the Th1-type cellular immune response in largemouth bass after immersion immunization. This indicates that the immunization strategy can coordinate innate and adaptive immunity to form a multi-layered immune defense, which may be the immune basis for achieving a 97.6% protection rate.

[0065] 3 Discussion Ginsenoside Rg1 can significantly enhance the immune effect of vaccines.

[22] These components enhance antigen presentation and activate immune cells, thereby promoting immune memory and antibody production. This study initially screened four effective extracts from traditional Chinese medicine—ginsenosides, β-glucan, astragalus polysaccharides, and anisodamine—as activators for freeze-dried vaccines. Through titer determination and immunoprotection experiments of the activated freeze-dried vaccine, ginsenosides were found to be the best in maintaining vaccine activity and enhancing immunogenicity. Further comparison of the activation effects of three main ginsenosides, Rg1, Rb1, and Ro, revealed that ginsenoside Rg1 was the optimal solvent for freeze-dried vaccines. Fish experiments confirmed that immersion immunization with freeze-dried vaccine activated with 20 mg / mL ginsenoside Rg1 resulted in a relative immunoprotection rate as high as 97.6%, significantly improving the vaccine's immunogenicity.

[0066] In summary, this invention, by screening the optimal freeze-dried vaccine activator, reducing vaccine loss during dissolution, optimizing the immersion immunization procedure, and increasing the amount of antigen uptake by fish during immersion immunization, can significantly enhance the immune protection effect. This establishes a highly efficient and stable immersion immunization procedure using a live attenuated freeze-dried largemouth bass rhabdovirus vaccine, significantly improving vaccine antigen uptake and immune response intensity, promoting the expression of related immune factors (especially IL-12), and significantly enhancing the immune response level of fish. The optimized immunization procedure in this study achieved a relative immune protection rate as high as 97.6%, providing an efficient prevention and control solution for largemouth bass rhabdovirus disease.

[0067] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. An activator for a largemouth bass rhabdovirus attenuated freeze-dried vaccine, characterized in that, Including ginsenoside Rg1.

2. The activator for the largemouth bass rhabdovirus attenuated freeze-dried vaccine according to claim 1, characterized in that, The activator of the largemouth bass rhabdovirus attenuated freeze-dried vaccine includes an aqueous solution of ginsenoside Rg1 with a concentration of 5-20 mg / mL.

3. Application of ginsenoside Rg1 in the preparation of attenuated freeze-dried vaccine of largemouth bass rhabdovirus.

4. The working concentration of ginsenoside Rg1 according to claim 3 is 5~20 mg / mL.

5. A method for activating the largemouth bass rhabdovirus attenuated freeze-dried vaccine, characterized in that, Includes the following steps: The largemouth bass rhabdovirus attenuated freeze-dried vaccine was dissolved in an aqueous solution of ginsenoside Rg1.

6. The method according to claim 5, characterized in that, The concentration of the ginsenoside Rg1 aqueous solution is 5~20 mg / mL.

7. The method according to claim 5, characterized in that, The largemouth bass rhabdovirus attenuated freeze-dried vaccine had a viral titer of 10 in the viral fluid before freeze-drying. 7.3 TCID 50 / mL.

8. The method according to claim 5, characterized in that, The virus strain in the largemouth bass rhabdovirus attenuated freeze-dried vaccine is largemouth bass rhabdovirus MSRV-0509, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:V202412.

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