Infectious spleen and kidney necrosis virus recombinant adenovirus oral preparation as well as preparation method and application thereof

Abstract

The invention relates to an infectious spleen and kidney necrosis virus recombinant adenovirus oral preparation which is characterized by comprising the following components in percentage by mass: 2-4% of bovine serum albumin, 3-7% of lactose, 1-3% of glycine, 1-3% of mannitol, 2-4% of povidone, 1-2% of talcum powder and 0.8-3% of magnesium stearate. The balance is the cultured infectious spleen and kidney necrosis virus recombinant adenovirus liquid. The infectious spleen and kidney necrosis virus recombinant adenovirus strain is used as a vaccine virus, a wrapping agent, a protective agent and the like are added and mixed, the mixture can be added into a conventional feed, vaccine oral inoculation is achieved, the immune effect is equivalent to vaccine injection inoculation, and the vaccine has the advantages of being convenient to use, saving labor cost, reducing fish body stress loss and the like. The oral preparation disclosed by the invention is good in stability, the loss rate of active substances is low within the period of validity, and the disintegration time limit accords with national regulations.

Description

Technical fields:

[0001] This invention belongs to the field of biotechnology, specifically relating to an oral protective agent for recombinant adenovirus of infectious spleen and kidney necrosis virus, its preparation method, and its application. Background technology:

[0002] Infectious spleen and kidney necrosis virus (ISKNV) is a typical representative species of the genus *Mammovirus* in the family Iridoviridae. This virus is prevalent in various farmed fish species, including mandarin fish, largemouth bass, grouper, large yellow croaker, and snakehead, and is characterized by high infectivity and mortality. It has a mortality rate of up to 100% in mandarin fish, making it one of the most serious viral diseases affecting farmed fish in my country and worldwide. For a long time, fish farmers have used large quantities of various chemical drugs and antibiotics to prevent this disease. However, with increasingly stringent requirements for the quality and safety of aquatic products in domestic and international consumer markets, drug residues have become a serious problem.

[0003] The immunization routes for fish vaccines can be broadly categorized into: injection immunization, immersion immunization, and oral immunization. Injection immunization involves injecting the vaccine into the fish via the peritoneal cavity or intramuscularly. The biggest advantage of injection immunization is the ability to precisely control the dosage, resulting in a high level of immune protection. However, injection immunization can cause damage to the fish's body surface and a strong stress response, which can affect the vaccine's effectiveness. Furthermore, injection immunization incurs high labor and production costs, making it unsuitable for large-scale, common economic fish farming and hindering its widespread adoption. Immersion immunization involves placing the fish in water containing the vaccine for a period of time, allowing the fish to absorb the vaccine through water exchange and osmotic pressure. However, the fish's own biological barriers make it difficult for the vaccine to penetrate the body, resulting in only a low level of immunity. Therefore, many researchers have developed suitable adjuvants to improve the effectiveness of immersion immunization. While immersion immunization provides some protection in the short term, the duration of protective antibody levels cannot cover the fish's growth cycle, and booster immunization is almost impossible in actual production, thus presenting significant drawbacks.

[0004] Oral immunization refers to the immunization route in which vaccines are mixed with feed or directly placed into the mouth of fish. Oral immunization generally does not cause harm to the fish and is suitable for immunizing large populations. However, after entering the fish's body, vaccines are first degraded or have their activity reduced by various digestive enzymes in the digestive tract, thus affecting the vaccine's immunization efficacy (Tang et al. 2017). Studies have shown that encapsulating vaccines with suitable materials can reduce the impact of the digestive tract on vaccine efficacy (Irie et al. 2003). Wang et al. (2018) prepared an oral vaccine by encapsulating the recombinant protein rSrr of Streptococcus dolphinus in alginate and chitosan composite microspheres. Immunization tests on spotted catfish showed that this oral vaccine could induce higher serum antibody levels and lysozyme activity, with a relative immunization protection rate of up to 60%. Embregts et al. prepared an oral vaccine based on a yeast oral vaccine delivery system, combining a model antigen and green fluorescent protein. Immunization experiments showed that the vaccine delivery process in the intestines of carp and rainbow trout could be observed using green fluorescent protein, triggering a rapid local innate immune response in the intestines, followed by a systemic immune response in the spleen. Bacillus subtilis has been frequently used in oral vaccine delivery systems for aquatic products in recent years. Jiang et al. (2019) constructed a Bacillus subtilis oral vaccine (CotC-VP4) against grass carp reovirus. Immunization experiments showed that CotC-VP4 induced higher serum antibody titers and protection against the virus in grass carp, with a relative immunoprotection rate of up to 47%, and induced effective innate and adaptive immunity. Studies have shown that recombinant antigen proteins prepared in nanoparticle form can withstand the intestinal environment of fish and still maintain high biological activity (Thwaite et al. 2018). For smaller fish, the palatability of feed particle size must be considered, and oral immunization is difficult to achieve the desired effect for some fish that eat live bait.

[0005] It is evident that all existing oral vaccines suffer from poor immunization efficacy. Summary of the Invention:

[0006] To address the problems in the prior art, this invention discloses an oral protective agent for recombinant adenovirus of infectious spleen and kidney necrosis virus. This invention also discloses the preparation method and its uses.

[0007] The technical solution of this invention is as follows:

[0008] A recombinant adenovirus solution for infectious spleen and kidney necrosis virus, wherein the preparation method of the viral solution is as follows: resuscitate and passage human embryonic kidney cells (HEK293), and when the HEK293 cell confluence reaches 80-95%, wash three times with PBS, according to 5×10 2 ~5×10 3virus / cm 2 Add the appropriate amount of infectious spleen and kidney necrosis virus recombinant adenovirus, incubate at 35-38℃ in a carbon dioxide incubator for 0.5-2 hours, then add DMEM maintenance medium containing 0.5-2% fetal bovine serum and continue culturing until more than 85% of the cells detach.

[0009] Furthermore, the recombinant adenovirus solution containing infectious spleen and kidney necrosis virus is prepared as follows: human embryonic kidney cells (HEK293) are resuscitated and passaged. When the HEK293 cells reach approximately 90% confluence, the cell culture supernatant is discarded, and the cells are washed three times with sterile PBS at a concentration of 1×10⁻⁶. 3 virus / cm 2 Add the appropriate amount of infectious spleen and kidney necrosis virus recombinant adenovirus, incubate at 37°C in a carbon dioxide incubator for 1 hour for adsorption, then add DMEM maintenance medium containing 1% fetal bovine serum and continue culturing until 90% of the cells detach, and the product is obtained.

[0010] A lyophilized preparation of recombinant adenovirus of infectious spleen and kidney necrosis virus (ISBN) comprises: recombinant adenovirus of ISBN and a lyophilization protectant, wherein the lyophilization protectant comprises: lactose, bovine serum albumin (BSA), mannitol, and glycine; the mass percentage of the recombinant adenovirus of ISBN and the lyophilization protectant is: 2-4% bovine serum albumin (BSA), 3-7% lactose, 1-3% glycine, 1-3% mannitol, and the balance being the recombinant adenovirus solution of ISBN.

[0011] Furthermore, in the lyophilized preparation of the infectious spleen and kidney necrosis virus recombinant adenovirus, the mass percentages of the infectious spleen and kidney necrosis virus recombinant adenovirus and the lyophilization protectant are: 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, and the remainder is infectious spleen and kidney necrosis virus recombinant adenovirus liquid.

[0012] The method for preparing the lyophilized preparation of the infectious spleen and kidney necrosis virus recombinant adenovirus involves mixing bovine serum albumin (BSA), lactose, glycine, mannitol, and infectious spleen and kidney necrosis virus recombinant adenovirus solution, dispensing the mixture, and then lyophilizing it in a lyophilizer to obtain the final product.

[0013] Furthermore, the freeze-drying method involves repeatedly freezing and thawing at -20±5℃ / 25±5-20℃ / 25℃ 1-5 times, preferably 3 times.

[0014] An oral preparation of recombinant adenovirus for infectious spleen and kidney necrosis virus comprises 2-4% bovine serum albumin (BSA), 3-7% lactose, 1-3% glycine, 1-3% mannitol, 2-4% povidone, 1-2% talc, 0.8-3% magnesium stearate, with the remainder being cultured recombinant adenovirus solution for infectious spleen and kidney necrosis virus.

[0015] Furthermore, the oral preparation of the infectious spleen and kidney necrosis virus recombinant adenovirus comprises 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, 3% povidone, 1.5% talc, 1% magnesium stearate, and the remainder being cultured infectious spleen and kidney necrosis virus recombinant adenovirus solution.

[0016] The preparation method of the oral preparation of the infectious spleen and kidney necrosis virus recombinant adenovirus includes the following steps: (i) placing the infectious spleen and kidney necrosis virus recombinant adenovirus solution at -20±5℃ / 25±5℃ and repeatedly freezing and thawing 1-5 times, preferably 3 times.

[0017] (ii) Bovine serum albumin (BSA), lactose, glycine, mannitol, and recombinant adenovirus of infectious spleen and kidney necrosis virus were mixed, dispensed, and freeze-dried into powder in a freeze dryer.

[0018] (iii) Store at 4℃ for later use;

[0019] (iv) Add povidone to the lyophilized powder and mix for 1-5 minutes, preferably 3 minutes. Then add talc and magnesium stearate and continue mixing for 5-15 minutes, preferably 10 minutes. The process temperature is 4-8°C.

[0020] Application of recombinant adenovirus liquid, lyophilized preparation, or oral formulation of infectious spleen and kidney necrosis virus in fish vaccines.

[0021] This invention uses a recombinant adenovirus strain of infectious spleen and kidney necrosis virus as the vaccine virus. After being mixed with an oral encapsulating agent, it can be added to regular feed for oral vaccination. The immunization effect is comparable to that of injection vaccination, offering advantages such as ease of use, reduced labor costs, and decreased stress losses in fish. The freeze-dried formulation and oral formulation described in this invention exhibit good stability, low loss rate of active substances within the shelf life, and disintegration time limits that meet national regulations. The freeze-dried formulation and oral formulation disclosed in this invention solve long-standing problems in the prior art, providing an effective protective agent and encapsulating agent to protect the activity of the viral vaccine and resist degradation by digestive enzymes. This makes oral vaccination of aquatic animals possible, significantly reducing labor costs and enhancing immunization, covering the animal's growth period. Detailed implementation method:

[0022] The present invention will be further described in detail below with reference to embodiments, but this is not intended to limit the scope of protection of the present invention. The reagents used in this embodiment can all be purchased commercially or prepared according to existing techniques.

[0023] Example 1: Preparation of Recombinant Adenovirus Vaccine against Infectious Splenone and Kidney Necrosis Virus

[0024] Step 1: Obtaining the main protective gene of ISKNV. The specific implementation is as follows:

[0025] (1) Design of PCR amplification primers

[0026] Referring to GenBank (https: / / www.ncbi.nlm.nih.gov / nuccore / MW883606.1): MW883606.1, the ISKNV sequence design mainly protects the amplification primers of the "DNA-binding protein" gene, and incorporates KpnI and NotI restriction sites.

[0027] Upstream primer: 5'-CAAAGATCTATGCATTGATTTTCAT-3'

[0028] Downstream primer: 5'-CTAGCGGCCGCGATGTCAGACAGGATAT-3'

[0029] (2) Gene amplification and recovery

[0030] The pMD18-T plasmid containing the "DNA-binding protein" gene was diluted 100-fold with double-distilled water. 1 μL was used as a template to amplify the target fragment. The PCR reaction system is as follows:

[0031]

[0032] PCR reaction conditions: 95℃ pre-denaturation for 5 min; 94℃ for 45 sec, 54℃ for 30 sec, 72℃ for 50 sec, for a total of 35 cycles; final extension at 72℃ for 10 min, and storage at 4℃; the PCR product was placed on a 0.8% agarose gel, electrophoresed at 60V for 20 min, and the fragment of the target band size was excised and the amplified product was recovered.

[0033] Step 2: Construction of the adenovirus shuttle vector pAdTrack. The specific implementation is as follows;

[0034] (1) Double digestion of the protective gene with Kpn I and Not I;

[0035] Add the following substances sequentially to a 1.5 mL Eppendorf tube:

[0036]

[0037] After incubating in a 37°C water bath for 6 hours, the gel containing the target band was excised and recovered by electrophoresis on an 8 g / L agarose gel.

[0038] (2) KpnI and NotI double digestion of adenovirus shuttle vector pAdTrack-CMV;

[0039] Add the following substances sequentially to a 1.5 mL Eppendorf tube:

[0040]

[0041] After incubating in a 37°C water bath for 6 hours, the gel containing the target band was excised and recovered by electrophoresis on an 8 g / L agarose gel.

[0042] (3) Ligation of the protective gene with the shuttle plasmid vector pAdTrack-CMV;

[0043] Connecting the reaction system:

[0044]

[0045]

[0046] 16℃ water bath overnight;

[0047] (4) Transformation and identification of the protective gene and the pAdTrack-CMV vector ligation product;

[0048] Take a tube of DH5α Escherichia coli competent cells and place it on an ice-water mixture. After it is completely thawed, add 10 μL of the ligation product to the tube, mix gently, incubate on ice for 30 min, then heat shock in a 42°C water bath for 90 s and incubate on ice for 3 min. Add 800 μL of LB medium, incubate at 37°C with shaking at 200-250 rpm for 1 h, then centrifuge at 8000 rpm for 1 min, discard part of the supernatant, and resuspend the precipitate in about 100 μL of the supernatant.

[0049] Spread 100 μL of the above mixture evenly on an LB agar plate containing kanamycin (50 μg / mL), and after it is completely dried, incubate at 37°C overnight (12–16 h); pick a single colony and place it in 3 mL of LB liquid medium containing 50 μg / mL kanamycin, and incubate at 37°C overnight; extract plasmids by alkaline lysis, and then perform enzyme digestion, PCR, and sequencing identification;

[0050] Step 3: Construction of the recombinant adenovirus backbone vector. The specific implementation is as follows;

[0051] (1) Linearize the recombinant shuttle vector obtained in step two by digestion with Pme I. Add each component as shown below:

[0052]

[0053] After being placed in a 37°C water bath for 6 hours, the sample was recovered by agarose gel electrophoresis.

[0054] (2) The linearized recombinant adenovirus shuttle vector was transformed into Escherichia coli BJ5183 competent cells containing the adenovirus backbone vector pAd-Easy-1, and the recombinant adenovirus backbone vector was screened out.

[0055] Step 4: Obtaining recombinant adenovirus expressing the ISKNV protective gene. The specific implementation is as follows:

[0056] The recombinant adenovirus backbone vector was linearized by Pac I digestion. The digestion reaction system was as follows:

[0057]

[0058] Add 2.5 volumes of anhydrous ethanol and 1 / 10 volume of 3 mol / L NaAc to the enzyme digestion product, precipitate at -20℃ for 1 h, centrifuge at 12000 r / min for 15 min at 4℃, discard the supernatant, wash once with 70% ethanol, air dry, and then add 20 μL H2O to dissolve the DNA.

[0059] (2) Obtaining recombinant adenovirus

[0060] The linearized recombinant adenovirus backbone vector was transfected into human embryonic kidney cells (HEK 293) for packaging and passage; the viral fluid harvested after HEK 293 cells showed stable lesions was the infectious spleen and kidney necrosis virus recombinant adenovirus vaccine.

[0061] Example 2: Preparation of an oral vaccine based on recombinant adenovirus of infectious spleen and kidney necrosis virus;

[0062] Step 1: Resuscitate and passage human embryonic kidney cells (HEK293) using standard methods. When the HEK293 cells reach approximately 90% confluence, discard the cell culture supernatant, wash three times with sterile PBS, and then repeat the process at a ratio of 1×10⁻⁶ cells / mL. 3 virus / cm 2 Add the amount of infectious spleen and kidney necrosis virus recombinant adenovirus, and after adsorption in a 37°C carbon dioxide incubator for 1 hour, add DMEM maintenance medium containing 1% fetal bovine serum and continue culturing. After 90% of the cells detach, place them in a -20°C / 25°C freeze-thaw cycle 3 times.

[0063] Step 2: Prepare the vaccine according to the following weight percentages: 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, and 87% recombinant adenovirus of infectious spleen and kidney necrosis virus. Mix well and dispense into 2ml / bottles, then freeze-dry into powdered vaccine in a freeze dryer.

[0064] Step 3, Testing of powdered freeze-dried vaccine: The freeze-dried vaccine was tested for sterility, mold, mycoplasma, safety and efficacy according to the testing methods specified in the "Veterinary Pharmacopoeia of the People's Republic of China". If all tests were passed, the vaccine was stored at 4℃ for later use.

[0065] Step 4: Add 3% povidone by weight to the freeze-dried vaccine powder and mix in a mixer for 3 minutes. Then add 1.5% talc and 1% magnesium stearate by weight and continue mixing for 10 minutes. The temperature of all operations is controlled at 4 to 8°C.

[0066] Step 5: After packaging the oral recombinant adenovirus vaccine powder for infectious spleen and kidney necrosis virus prepared in Step 4, it can be stored for a long time at -15℃. When using, use 1g of vaccine powder per 10g of fish body weight, and mix the vaccine powder evenly with the feed. If using pelleted feed, you can spray some water during mixing to help the vaccine powder adhere to the pellets.

[0067] Example 3: Optimization and Selection of Lyophilization Protectants

[0068] To verify the protective effect of the lyophilized protectant against the recombinant adenovirus vaccine against infectious spleen and kidney necrosis virus, the following control trial was conducted:

[0069] Table 1: Optimization and Selection of Lyophilization Protectants

[0070]

[0071] Based on the components in the table, the virus lyophilized powder was harvested according to steps one and two in Example 2, then reconstituted with the original volume, and subjected to TCID. 50 Measurement. The specific measurement method is as follows.

[0072] HEK293 cells were cultured in 96-well cell culture plates until 90% confluence was achieved. Recombinant virus was serially diluted 10-fold, and 100 μL of virus solution was inoculated into each well. Each dilution was replicated in 8 wells, with a blank control included. Cells were incubated at 37°C for 1 h using CO2, then the supernatant was discarded. 100 μL of LMEM maintenance medium was added, and the cells were cultured for another 48 hours. The supernatant was then discarded, and the cells were washed twice with PBS. 100 μL of mouse anti-human type 5 replication-defective adenovirus polyclonal antibody (1:100 dilution) was added to each well, and the cells were incubated in a humidified chamber at 37°C for 2 h. The primary antibody was discarded, and the cells were washed three times with PBS. 100 μL of goat anti-mouse IgG secondary antibody (1:2500 dilution) was added to each well, and the cells were incubated at 37°C for 45 min. The secondary antibody was discarded, and the cells were washed three times with PBS. The cells were then observed under a fluorescence microscope, and the TCID was calculated using the Reed-Muench method. 50 The results are shown in the table below, where the potency of step one in Example 2 is 10. 8.3 TCID 50 / mL.

[0073] Table 2: Virus titer detection results of different freeze-drying protectants

[0074]

[0075] Based on the titer determination results above, it is evident that the lyophilization protectant selected in this invention provides better protection compared to the lyophilization protectants in Comparative Examples 1-4. Although the number of selected protective components increased by 0.5 times when using the protectant described in Example 2, the virus titer was slightly lower than that in Example 1, while the input cost actually increased. In Example 3, reducing the content of each component by 0.5 times resulted in a protective effect significantly improved by two orders of magnitude. The results of Examples 2 and 3 indicate that both adding more and less protective components affect the virus protection effect. Using the lyophilization protectants in Comparative Examples 1-4, regardless of which protective component was missing, the virus titer differed from that in Example 1 by three orders of magnitude. This demonstrates that the amount of each component in the lyophilization protectant used in this invention is particularly important for the lyophilization protection effect.

[0076] Example 4: Optimization and Selection of Encapsulating Agents

[0077] Bioactive substance encapsulation agents (embedding agents / carriers) are key materials used to protect active ingredients, control release, and improve bioavailability. Based on their source and structure, they can be divided into: natural polymers, such as chitosan, sodium alginate, starch, gelatin, pullulan, etc. These materials have good biocompatibility and are biodegradable, but their mechanical / barrier properties need modification and enhancement, making them unsuitable for vaccine encapsulation; and synthetic polymers, such as epoxy resins (Epon812, Spurr) and acrylic resins (LR). Materials like White and Lowicryl offer good permeability and low-temperature polymerization capabilities, but some exhibit toxicity or volume shrinkage issues, making them unsuitable for vaccine encapsulation. Lipid and cell membrane-based encapsulants, such as liposomes and erythrocyte membrane-coated micelles (e.g., ICG-PCL-Soluplus-TPGS), while possessing biomimetic structures that enhance targeting and prolong circulation time for multi-drug co-loading, are prone to causing immune side effects after entering the body, thus also unsuitable for vaccine encapsulation. Other applicable encapsulants are industrially used and all exhibit cytotoxic effects on vaccines. Furthermore, even ideal encapsulants like "liquid-liquid phase separation aggregates" are limited by their high cost, hindering widespread application. This invention, based on screening several encapsulants, yields an encapsulant component that ensures vaccine activity while saving production costs and enabling large-scale application. However, different component ratios can affect vaccine activity. To verify the protective effect of the encapsulant against recombinant adenovirus vaccines against infectious spleen and kidney necrosis virus, the following control experiment was conducted:

[0078] Table 3: Screening of different encapsulating agent components

[0079]

[0080]

[0081] Based on the components in the table, after harvesting the virus lyophilized powder according to step two of Example 2, it was reconstituted with the original volume and subjected to TCID. 50 Measurement. The specific measurement method is as follows.

[0082] HEK293 cells were cultured in 96-well cell culture plates until 90% confluence was achieved. Recombinant virus was serially diluted 10-fold, and 100 μL of virus solution was inoculated into each well. Each dilution was replicated in 8 wells, with a blank control included. Cells were incubated at 37°C for 1 h using CO2, then the supernatant was discarded. 100 μL of LMEM maintenance medium was added, and the cells were cultured for another 48 hours. The supernatant was then discarded, and the cells were washed twice with PBS. 100 μL of mouse anti-human type 5 replication-defective adenovirus polyclonal antibody (1:100 dilution) was added to each well, and the cells were incubated in a humidified chamber at 37°C for 2 h. The primary antibody was discarded, and the cells were washed three times with PBS. 100 μL of goat anti-mouse IgG secondary antibody (1:2500 dilution) was added to each well, and the cells were incubated at 37°C for 45 min. The secondary antibody was discarded, and the cells were washed three times with PBS. The cells were then observed under a fluorescence microscope, and the TCID was calculated using the Reed-Muench method. 50 The results are shown in the table below, where the potency of step one in Example 2 is 10. 8.3 TCID 50 / mL.

[0083]

[0084] Based on the titer determination results above, it is evident that the encapsulating agent composition ratio selected in Example 2 of this invention provides better protection than encapsulating agents with other composition ratios. In Example 1, reducing the amount of povidone by half resulted in a one-order-of-magnesium decrease in viral titer, so this method was not adopted. In Example 3, increasing the amount of povidone by half did not significantly reduce the viral titer, but it was slightly lower than the viral titer in Example 2. This would not only increase costs but also lead to a decrease in the titer per unit volume of oral vaccine, so this method was not adopted. In Examples 4 and 5, halving or increasing the amount of talc by half resulted in a one-order-of-magnesium decrease in viral titer, indicating that too little or too much talc would lead to viral inactivation, so this method was not adopted. In Examples 6 and 7, halving or increasing the amount of magnesium stearate by half would also lead to viral inactivation, so this method was also not adopted. This demonstrates that the amount of each component in the encapsulating agent used in this invention is particularly important for the protective effect against the virus.

[0085] Example 5: Evaluation of the Immunization Efficacy of Oral Vaccine Based on Recombinant Adenovirus of Infectious Splenone and Kidney Necrosis Virus

[0086] Experimental animals: Healthy mandarin fish (6.0±0.5g) were purchased from a fish farm in Jiangmen City, Guangdong Province. The water temperature was maintained at 28±1℃, dissolved oxygen was kept above 6mg / L, and the fluorescent lamps were set to a 12h / d light cycle. The fish were fed regularly each day, and uneaten food and feces were removed from the bottom of the tank. The water was changed every 3 days. The experiment was conducted after a 14-day period of temporary rearing.

[0087] Experimental animal grouping: The experimental mandarin fish were randomly divided into 6 groups (A, B, C, D, E, and F), with 20 fish in each group. Group A received the commercially available ISKNV inactivated vaccine according to the instructions; Group B received the oral vaccine against infectious spleen and kidney necrosis virus (ISBN) recombinant adenovirus; Group C received the ISBN recombinant adenovirus injection; Group D received the ISBN recombinant adenovirus immersion; Group E was the virus-positive control group; and Group F was the virus-negative control group.

[0088] Challenge experiment: 21 days after immunization, each group was intraperitoneally injected with 0.2 ml of ISKNV virus solution at a concentration of 10 times the median lethal dose (LD50), with the water temperature controlled at 28±1℃. Disease incidence was checked and recorded daily for 28 consecutive days. Mortality rates were calculated, and the relative percentage survival (RPS) was determined. RPS = (1 - % mortality rate in the immunized group / % mortality rate in the control group) × 100.

[0089] Results observation: 28 days after challenge, all fish in the positive control group (Group E) died, while all fish in the negative control group (Group F) survived, proving the experiment was successful. Table 4 shows that the recombinant adenovirus vaccine against infectious spleen and kidney necrosis virus prepared as an oral vaccine using this invention has no difference in immunization efficacy compared to injection or immersion administration.

[0090] Table 4 Results of the immune challenge experiment

[0091]

[0092] Example 6: A recombinant adenovirus solution for infectious spleen and kidney necrosis virus. The preparation method of the viral solution is as follows: resuscitate and passage human embryonic kidney cells (HEK293). When the HEK293 cell confluence reaches about 90%, discard the cell culture supernatant, wash three times with sterile PBS, and repeat the process at a ratio of 1×10⁻⁶. 3 virus / cm 2 The appropriate amount of recombinant adenovirus containing infectious spleen and kidney necrosis virus was added, and after adsorption in a 37°C carbon dioxide incubator for 1 hour, DMEM maintenance medium containing 1% fetal bovine serum was added and cultured for another 1 hour. Once 90% of the cells had detached, the product was obtained. It is used for fish vaccines.

[0093] Example 7: A lyophilized preparation of recombinant adenovirus of infectious spleen and kidney necrosis virus, comprising recombinant adenovirus of infectious spleen and kidney necrosis virus and a lyophilization protectant, wherein the lyophilization protectant comprises: lactose, bovine serum albumin, mannitol, and glycine; the mass percentage of the recombinant adenovirus of infectious spleen and kidney necrosis virus and the lyophilization protectant is: 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, and the balance being recombinant adenovirus of infectious spleen and kidney necrosis virus liquid.

[0094] The preparation of the lyophilized preparation of recombinant adenovirus of infectious spleen and kidney necrosis virus involves mixing bovine serum albumin (BSA), lactose, glycine, mannitol, and recombinant adenovirus of infectious spleen and kidney necrosis virus, dispensing the mixture, and then lyophilizing it in a lyophilizer.

[0095] Example 8: A lyophilized preparation of recombinant adenovirus of infectious spleen and kidney necrosis virus, comprising recombinant adenovirus of infectious spleen and kidney necrosis virus and a lyophilization protectant, wherein the lyophilization protectant comprises: lactose, bovine serum albumin, mannitol, and glycine; the mass percentage of the recombinant adenovirus of infectious spleen and kidney necrosis virus and the lyophilization protectant is: 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, and the remainder is recombinant adenovirus of infectious spleen and kidney necrosis virus solution.

[0096] The preparation of a lyophilized formulation of recombinant adenovirus containing infectious spleen and kidney necrosis virus involves mixing bovine serum albumin (BSA), lactose, glycine, mannitol, and recombinant adenovirus solution, aliquoting, and lyophilizing in a freeze dryer. The lyophilization process involves repeated freeze-thaw cycles at -20℃ and 25℃ three times to obtain the final product. This formulation is intended for use as a fish vaccine.

[0097] Example 9: An oral preparation of recombinant adenovirus for infectious spleen and kidney necrosis virus, comprising 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, 3% povidone, 1.5% talc, 1% magnesium stearate, and the remainder being cultured recombinant adenovirus solution for infectious spleen and kidney necrosis virus.

[0098] The preparation method is as follows: Step (I) The recombinant adenovirus solution of infectious spleen and kidney necrosis virus is placed at -20℃ / 25℃ and repeatedly frozen and thawed 3 times;

[0099] (ii) Bovine serum albumin (BSA), lactose, glycine, mannitol, and recombinant adenovirus of infectious spleen and kidney necrosis virus were mixed, dispensed, and freeze-dried into powder in a freeze dryer.

[0100] (iii) Store at 4℃ for later use;

[0101] (iv) Add povidone to the lyophilized powder and mix for 3 minutes. Then add talc and magnesium stearate and continue mixing for 10 minutes. The temperature of the process is 4-8℃.

[0102] Used for fish vaccines.

[0103] Example 10: Application of oral administration of recombinant adenovirus of infectious spleen and kidney necrosis virus in fish vaccine. The oral administration prepared in Example 9 is used at a dosage of 1g of oral administration per 10g of fish body weight. The oral administration is mixed evenly with the feed.

[0104] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A recombinant adenovirus fluid containing infectious spleen and kidney necrosis virus, characterized in that: The method for preparing the viral fluid is as follows: revive and passage human embryonic kidney cells (HEK293). When the HEK293 cell confluence reaches 80-95%, wash three times with PBS, and then repeat the process at a ratio of 5 × 10⁻⁶ cells / mL. 2 ~5×10 3 virus / cm 2 Add the appropriate amount of infectious spleen and kidney necrosis virus recombinant adenovirus, incubate at 35-38℃ in a carbon dioxide incubator for 0.5-2 hours, then add DMEM maintenance medium containing 0.5-2% fetal bovine serum and continue culturing until more than 85% of the cells detach.

2. The recombinant adenovirus fluid containing infectious spleen and kidney necrosis virus as described in claim 1, characterized in that: The method for preparing the viral fluid is as follows: Resuscitate and passage human embryonic kidney cells (HEK293). When the HEK293 cells reach approximately 90% confluence, discard the cell culture supernatant, wash three times with sterile PBS, and repeat the process at a ratio of 1×10⁻⁶. 3 virus / cm 2 Add the appropriate amount of infectious spleen and kidney necrosis virus recombinant adenovirus, incubate at 37°C in a carbon dioxide incubator for 1 hour for adsorption, then add DMEM maintenance medium containing 1% fetal bovine serum and continue culturing until 90% of the cells detach, and the product is obtained.

3. A lyophilized preparation of recombinant adenovirus for infectious spleen and kidney necrosis virus, characterized in that... include: The recombinant adenovirus of infectious spleen and kidney necrosis virus and a lyophilization protectant, wherein the lyophilization protectant comprises: lactose, bovine serum albumin, mannitol, and glycine; the mass percentage of the recombinant adenovirus of infectious spleen and kidney necrosis virus and the lyophilization protectant is: 2-4% bovine serum albumin (BSA), 3-7% lactose, 1-3% glycine, 1-3% mannitol, and the balance being the recombinant adenovirus solution of infectious spleen and kidney necrosis virus.

4. The lyophilized preparation of recombinant adenovirus for infectious spleen and kidney necrosis virus as described in claim 3, characterized in that: The recombinant adenovirus of infectious spleen and kidney necrosis virus and the lyophilization protectant have the following mass percentages: 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, and the remainder is the recombinant adenovirus solution of infectious spleen and kidney necrosis virus.

5. The method for preparing the lyophilized preparation of recombinant adenovirus of infectious spleen and kidney necrosis virus as described in claim 3, characterized in that: Bovine serum albumin (BSA), lactose, glycine, mannitol, and recombinant adenovirus of infectious spleen and kidney necrosis virus were mixed, dispensed, and freeze-dried in a freeze dryer to obtain the final product.

6. The preparation method according to claim 5, characterized in that: The freeze-drying method involves repeatedly freezing and thawing at -20±5℃ / 25±5-20℃ / 25℃ 1-5 times, preferably 3 times.

7. An oral preparation of recombinant adenovirus for infectious spleen and kidney necrosis virus, characterized in that... It includes 2-4% bovine serum albumin (BSA), 3-7% lactose, 1-3% glycine, 1-3% mannitol, 2-4% povidone, 1-2% talc, 0.8-3% magnesium stearate, and the balance is cultured recombinant adenovirus of infectious spleen and kidney necrosis virus.

8. The oral preparation of recombinant adenovirus for infectious spleen and kidney necrosis virus as described in claim 7, characterized in that... It includes 3% bovine serum albumin (BSA), 6% lactose, 2% glycine, 2% mannitol, 3% povidone, 1.5% talc, 1% magnesium stearate, and the remainder is cultured recombinant adenovirus of infectious spleen and kidney necrosis virus.

9. The method for preparing the oral recombinant adenovirus preparation of infectious spleen and kidney necrosis virus as described in claim 7, characterized in that: The steps include: (i) placing the recombinant adenovirus solution of infectious spleen and kidney necrosis virus at -20±5℃ / 25±5℃ for repeated freeze-thaw cycles 1-5 times, preferably 3 times; (ii) Bovine serum albumin (BSA), lactose, glycine, mannitol, and recombinant adenovirus of infectious spleen and kidney necrosis virus were mixed, dispensed, and freeze-dried into powder in a freeze dryer. (iii) Store at 4℃ for later use; (iv) Add povidone to the lyophilized powder and mix for 1-5 minutes, preferably 3 minutes. Then add talc and magnesium stearate and continue mixing for 5-15 minutes, preferably 10 minutes. The process temperature is 4-8°C.

10. Application of oral recombinant adenovirus preparation of infectious spleen and kidney necrosis virus in fish vaccines.

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