Fish nervous necrosis virus blocking protein, its preparation method and application

Abstract

The present application belongs to the field of prevention and control of aquatic pathogenic microorganisms, and particularly relates to a fish nervous necrosis virus blocking protein as well as a preparation method and application thereof. Due to the special structure, the protein can specifically bind to the receptors on the surface of fish nervous necrosis virus particles, so that the fish nervous necrosis virus loses the ability to invade fish cells, thereby blocking the fish nervous necrosis virus from infecting the host cells of fish, and avoiding the replication and proliferation of the fish nervous necrosis virus in the host. The fish nervous necrosis virus blocking protein provided by the present application has a high anti-nervous necrosis virus effect. The biological preparation product prepared by using the fish nervous necrosis virus blocking protein as an active material can significantly improve the anti-nervous necrosis virus ability of fish fry by using the oral method, is safe and pollution-free, can be applied on a large scale, provides a new way for the prevention and control of fish nervous necrosis virus disease, and further improves the economic benefits of fish culture.

Description

Technical Field

[0001] This invention belongs to the field of aquatic pathogenic microorganism control, specifically relating to a fish nerve necrosis virus blocking protein and its preparation method and application. Background Technology

[0002] In recent years, the aquaculture industry has developed rapidly, but the frequent outbreaks of fish nerve necrosis virus disease have severely restricted its healthy development. Fish nerve necrosis virus (NNV) belongs to the Nodaviridae family and the Betanodaviridae genus. It is a highly contagious RNA virus that can infect various marine and freshwater fish, especially juvenile fish. It primarily infects the central nervous system, retina, and brain of marine fish, leading to severe neurodegenerative diseases with extremely high mortality rates, causing huge economic losses to the aquaculture industry.

[0003] Currently, control measures for fish nerve necrosis virus disease are very limited. Although vaccination is a commonly used method to prevent infection, vaccines for fish nerve necrosis virus disease are still in the early stages of development, and their effectiveness against fish fry with underdeveloped immune systems is relatively limited. Therefore, developing safe and effective antiviral agents against fish nerve necrosis virus is of significant practical importance. Summary of the Invention

[0004] To address the problems in existing technologies, this invention provides a fish nerve necrosis virus (NSNV) blocking protein. Due to its unique structure, this protein can specifically bind to receptors on the surface of NNSV particles, rendering them incapable of infecting fish cells. This blocks NNSV infection of host cells, preventing its replication and proliferation within the host. The NNSV blocking protein provided by this invention exhibits highly efficient anti-NSNV activity. Biological agents prepared using this protein as the active material, administered orally via feed mixing, can significantly improve the resistance of fish fry to NNSV. Furthermore, it is safe and pollution-free, enabling large-scale industrial application and providing a new approach to the prevention and control of fish NNSV disease, thereby improving the economic benefits of fish farming.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a fish nerve necrosis virus blocking protein, characterized in that: the amino acid sequence of the fish nerve necrosis virus blocking protein is shown in SEQ ID NO.1.

[0007] A second aspect of the present invention provides a gene encoding the above-mentioned fish nerve necrosis virus blocking protein, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0008] A third aspect of the present invention provides a method for preparing the above-mentioned fish nerve necrosis virus blocking protein, comprising: using the encoding gene in... Bam HI and Hin dⅢ was ligated to the vector Pet-30a(+) as an enzyme cleavage site to obtain a recombinant plasmid; the recombinant plasmid was transformed into Escherichia coli BL21-DE3, and the expression of the target protein was induced by IPTG. After purification, the fish nerve necrosis virus blocking protein was obtained.

[0009] The fourth aspect of the present invention provides a feed premix additive for preventing fish nerve necrosis virus, comprising, by weight parts, 1-2 parts of the above-mentioned fish nerve necrosis virus blocking protein, 350-360 parts of basic excipients and 50-60 parts of plant extract excipients.

[0010] Preferably, by weight, it includes 1 part of the fish nerve necrosis virus blocking protein, 350 parts of basic excipients, and 50 parts of plant extract excipients.

[0011] Furthermore, the basic auxiliary materials include the following raw materials in parts by weight: 2-5 parts calcium-based bentonite, 2-4 parts modified zeolite powder, 0.3-0.8 parts potassium silicate, 3-4 parts onion powder, 10-20 parts soybean flour, 4-8 parts yeast powder, 8-25 parts rice bran, 3-6 parts fish oil, 8-25 parts egg white powder, and 8-16 parts soybean meal.

[0012] The particle size of the basic auxiliary materials is above 100 mesh.

[0013] Calcium-based bentonite is used as a mineral supplement.

[0014] Modified zeolite powder can be used as an adsorbent to improve the intestinal environment.

[0015] Potassium silicate, as a dispersant, promotes the mutual dispersion of various raw materials.

[0016] Onion powder has antibacterial and immune-enhancing effects.

[0017] Soy flour and yeast powder serve as sources of protein and B vitamins.

[0018] Rice bran, as a source of fiber.

[0019] Fish oil, as an energy source.

[0020] Egg white powder and soybean meal are used as protein sources.

[0021] Furthermore, the plant extract excipients include the following raw materials in parts by weight: Astragalus powder 0.5-1 part, Large-leaved Ivy 0.5-1 part, Houttuynia cordata 0.5-1 part, Viola yedoensis 0.5-1 part, Isatis indigotica 0.5-1 part, Chrysanthemum indicum 0.5-1 part, Artemisia argyi 0.1-0.8 part, Fennel 0.1-0.8 part, and Platycodon grandiflorus 0.8-2.5 parts.

[0022] Among them, the particle size of the plant extract excipients is above 100 mesh.

[0023] Astragalus, large-leafed chrysanthemum, houttuynia cordata, violet, isatis root, and wild chrysanthemum are natural antiviral and immune enhancers.

[0024] Mugwort and fennel have flavoring properties and are also beneficial to health.

[0025] Platycodon grandiflorus has immunomodulatory effects.

[0026] The fifth aspect of the present invention provides a method for preparing the above-mentioned feed premix additive for preventing fish nerve necrosis virus: the above-mentioned fish nerve necrosis virus blocking protein, basic excipients and plant extract excipients are added to a mixer in sequence and stirred evenly by the mixer to obtain the feed premix additive for preventing fish nerve necrosis virus.

[0027] The sixth aspect of the present invention provides the application of the above-mentioned fish nerve necrosis virus blocking protein or the above-mentioned feed premix additive for preventing fish nerve necrosis virus in the preparation of feed for preventing fish nerve necrosis virus.

[0028] Furthermore, the feed premix additive for preventing fish nerve necrosis virus is added to the feed for preventing fish nerve necrosis virus at a mass percentage of 0.1% to 1%.

[0029] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0030] The fish nerve necrosis virus blocking protein provided by this invention can specifically bind to receptors on the surface of fish nerve necrosis virus particles, thereby preventing them from infecting fish cells. This blocks the infection of fish host cells by the nerve necrosis virus and prevents it from replicating and multiplying in the host, achieving a highly efficient anti-nerve necrosis virus effect. When this protein is added to feed premix additives, it can significantly improve the body's anti-nerve necrosis virus ability. It is also safe and pollution-free, and can be applied on a large scale in the industry, thereby improving the economic benefits of fish farming. Attached Figure Description

[0031] Figure 1 This is a schematic diagram showing the results of induction of the target protein expression by the fish nerve necrosis virus blocking protein of the present invention;

[0032] Figure 2 This is a schematic diagram of the SDS-PAGE results of the purified fish nerve necrosis virus blocking protein of the present invention. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0034] Unless otherwise specified, all materials, reagents, and experimental equipment involved in the embodiments of this invention are commercially available products.

[0035] Example 1

[0036] A fish nerve necrosis virus blocking protein, with its amino acid sequence (SEQ ID NO.1):

[0037] GGGGGSRRFPPLPAVIEEPMETDGGGGSGGGGGSGGGGSMVRKGEKKLAKPGGGGSGTTKAANPQPRRRGGGGSGANNRRRSNRTDAPVSGGGGSGLPDPTDNDHTFGGGGSGWESRTVRPQYTRGGGGSGSSGKEQRLTS PGRGGGGSGLETPEETTAGGGGSGDSLSTNDFKSGGGGSGLGTGDVDRAVYWSGGGGSGkkkkWDNFNKTFTDSGGGGSGYYSDEQPRQILLGGGGSGVGTVCTRVDSENGGGGSGKKKKNNRRRSNRTDAPGGGGSGIEGR

[0038] Gene sequence optimized for expression codons in E. coli (SEQ ID NO.2):

[0039] GGTGGTGGTGGTTCTGGTTCTCGTCGTTTCCCGCCGCTGCCGGCTGTTATCGAAGAACCGATGGAAACCGACGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTATGGTTCGTAAAGGTGAAAAAAAACTGGCTAAACCGGGTGGTGGTGGTTCTGGTACCACCAAAGCTGCTAACCCGCAGCCGCGTCGTCGTGGTGGTGGTGGTTCTGGTGCTAACAACCGTCGTCGTTCTAACCGTACCGACGCTCCGGTTTCTGGTGGTGGTGGTTCTGGTCTGCCGGACCCGACCGACAACGACCACACCTTCGGTGGTGGTGGTTCTGGTTGGGAATCTCGTACCGTTCGTCCGCAGTACACCCGTGGTGGTGGTGGTTCTGGTTCTTCTGGTAAAGAACAGCGTCTGACCTCTCCGGGTCGTGGTGGTGGTGGTTCTGGTCTGGAAACCCCGGAAGAAACCACCGCTGGTGGTGGTGGTTCTGGTGACTCTCTGTCTACCAACGACTTCAAATCTGGTGGTGGTGGTTCTGGTCTGGGTACCGGTGACGTTGACCGTGCTGTTTACTGGTCTGGTGGTGGTGGTTCTGGTAAAAAAAAAAAATGGGACAACTTCAACAAAACCTTCACCGACTCTGGTGGTGGTGGTTCTGGTTACTACTCTGACGAACAGCCGCGTCAGATCCTGCTGGGTGGTGGTGGTTCTGGTGTTGGTACCGTTTGCACCCGTGTTGACTCTGAAAACGGTGGTGGTGGTTCTGGTAAAAAAAAAAAAAACAACCGTCGTCGTTCTAACCGTACCGACGCTCCGGGTGGTGGTGGTTCTGGTATCGAAGGTCGT

[0040] The preparation method of the fish nervous necrosis virus blocking protein is as follows:

[0041] Based on the above gene sequence, restriction enzyme sites BamHI and HindⅢ were introduced and synthesized by Shanghai Sangon Biotech Co., Ltd. These sites were cloned and ligated to the corresponding restriction sites of the vector pET-30a(+) using the restriction enzyme sites at both ends of the gene sequence. The cloned strain was transformed into *E. coli* DH5α using the heat shock method. The transformed strain was plated on LB solid medium containing 50 μg / ml Kan+ and grown. Transformants containing the recombinant plasmid were screened by colony PCR and inoculated into LB liquid medium containing 50 μg / ml Kan+ at 37°C with shaking at 200 rpm for 12 hours. The recombinant plasmid was then extracted.

[0042] The recombinant plasmid containing the target gene fragment was transformed into Escherichia coli BL21(DE3) competent cells by heat shock method. The transformed cells were plated on LB solid medium containing 50 μg / ml Kan+ and screened for BL21(DE3) transformants containing the recombinant plasmid.

[0043] Single colonies of BL21(DE3) bacteria containing recombinant plasmids, grown on Kan+ resistant plates, were picked with sterile toothpicks and inoculated into 10 ml of LB broth containing 50 μg / ml Amp+. The culture was incubated overnight at 37°C and 200 rpm with constant temperature shaking. The next day, the bacterial culture was transferred to LB broth containing 50 μg / ml Amp+ at a ratio of 1:100. After culturing until the OD600 value reached 0.5 (3 hours), IPTG stock solution (100 mmol / L) was added at a ratio of 1:200, and the culture was continued for 5 hours to induce the expression of the target protein. The induction of the target protein by the fish nerve necrosis virus blocking protein was verified as follows. Figure 1 As shown (M: 180kDa color-stained pre-stained protein molecular weight standard; Lane 1: bacterial culture without IPTG induction; Lanes 2-3: bacterial culture induced by IPTG).

[0044] Purification of fish nerve necrosis virus blocking protein:

[0045] The *E. coli* fermentation broth expressing the recombinant protein was centrifuged at 5000 rpm for 8 minutes to collect bacterial cells. The cells were resuspended in PBS (1 / 20 volume of fermentation broth) and homogenized using a high-pressure homogenizer, with protease inhibitors added during the homogenization process. The homogenized liquid mixture was centrifuged again at 13000 rpm, 4°C, for 10 minutes, and the precipitate was collected. The target protein (precipitate) was resuspended in PBS containing 0.2% Triton-X100 and 0.6M urea, and centrifuged at 10000 rpm for 20 minutes at room temperature to collect the precipitate. This washing process was repeated twice to remove most of the soluble impurities. The washed precipitate was dissolved in PBS containing 8M urea, and centrifuged at room temperature for 20 minutes until the precipitate was completely dissolved and free of white particles. The precipitate was then centrifuged at 13000 rpm, 4°C to remove cell debris and unbroken cells, and the supernatant was collected to obtain the purified protein. The SDS-PAGE validation image of the purified fish nerve necrosis virus blocking protein is shown below. Figure 2 As shown (M: 180kDa color-stained pre-stained protein molecular weight standard; Lane 1: protein solution before purification; Lane 2: protein washing solution; Lane 3: purified protein solution from collection 1; Lane 4: purified protein solution from collection tube 2; Lane 5: purified protein solution from collection tube 3), the obtained purified protein was spray-dried and stored at 5℃ for later use.

[0046] Example 2

[0047] This embodiment provides a feed premix additive for preventing fish nerve necrosis virus:

[0048] Basic auxiliary materials (particle size 100 mesh): 3 parts calcium-based bentonite, 2 parts modified zeolite powder, 0.5 parts potassium silicate, 3 parts onion powder, 10 parts soybean powder, 4 parts yeast powder, 10 parts rice bran, 5 parts fish oil, 10 parts egg white powder, and 12 parts soybean meal.

[0049] Plant extract excipients (particle size 100 mesh): Astragalus powder 0.5 parts, large-leafed green 0.5 parts, houttuynia cordata 0.5 parts, violet 0.5 parts, isatis root 0.5 parts, wild chrysanthemum 0.5 parts, mugwort 0.3 parts, fennel 0.3 parts, and platycodon grandiflorus 2 parts.

[0050] One part of fish nerve necrosis virus blocking protein, 350 parts of basic excipients and 50 parts of plant extract excipients were added to a mixer in sequence and mixed thoroughly to obtain a feed premix additive for fish nerve necrosis virus.

[0051] Example 3

[0052] Evaluation of the efficacy of blocking fish nerve necrosis virus:

[0053] Case 1:

[0054] To verify the ability of fish nerve necrosis virus blocking protein to block nerve necrosis virus (NNV) infection at the cellular level.

[0055] Experimental materials

[0056] Cell line: Snakehead fish cell line (SSN-1);

[0057] Virus: Neuronecrosis Virus (NNV);

[0058] Blocking protein: Purified neuronecrosis virus blocking protein (NNV-BP), sterilized by filtration through a 0.22μm pinhole filter;

[0059] Culture medium: Leibovitz's L-15 medium;

[0060] Other reagents: PBS buffer, trypsin, fetal bovine serum (FBS), antibiotics (such as penicillin-streptomycin).

[0061] Experimental steps

[0062] A. SSN-1 cells were cultured in L-15 medium containing 10% FBS and 1% antibiotics at 37°C and 5% CO2. Once a monolayer of cells had grown in the cell culture flask, the cells were digested with trypsin and passaged.

[0063] B. Seed cells into 12-well cell culture plates, 2 × 10⁶ cells per well. 5 Infection experiments were conducted on cells cultured for approximately 48 hours until over 90% of the cells adhered to the culture vessel.

[0064] C. Fish nerve necrosis virus blocking protein was diluted to different concentrations (0 µg / ml, 0.1 µg / ml, 1 µg / ml, 10 µg / ml) in serum-free L-15 medium. These were labeled sequentially as the infection control group, low-concentration experimental group, medium-concentration experimental group, and high-concentration experimental group.

[0065] D. Add different concentrations of fish nerve necrosis virus blocking protein to SSN-1 cells cultured in 12-well cell culture plates, with 3 parallel wells for each concentration, and incubate at 37°C for 1 hour.

[0066] E.NNV cell culture virus solution was centrifuged to remove cell debris, and the virus solution was diluted with serum-free L-15 medium.

[0067] After incubation with F. nerve necrosis virus blocking protein, aspirate the culture medium, add medium containing NNV (MOI=1), and incubate at 37°C for 1 hour. Aspirate the virus solution, wash the cells three times with PBS, allowing them to stand for 3-5 minutes each time, and add fresh complete culture medium to continue culturing.

[0068] G. Cells were collected 24 hours after infection, and uninfected cells were used as a blank control group. Total RNA was extracted from cell samples of each group, reverse transcribed into cDNA, and viral load levels of each group were detected by qPCR.

[0069] The results are shown in Table 1:

[0070] Table 1. Virus load in cells infected with nerve necrosis virus after treatment with different concentrations of nerve necrosis virus blocking protein.

[0071]

[0072] Table 1 shows that the CT value for NNN virus detection in the infected control group (without NNN virus blocking protein treatment) was 21.03, indicating successful viral infection and replication. The CT value for NNN virus detection in the low-concentration experimental group was 28.52, in the medium-concentration experimental group it was 32.45, and in the high-concentration experimental group there was no CT value. The blank cell control also had no CT value and served as a valid negative control. Therefore, as the concentration of NNN virus blocking protein increases, the CT value for NNN virus detection increases, and the viral load decreases. High-concentration blocking protein may effectively block NNN virus infection.

[0073] Experimental results showed that fish nerve necrosis virus blocking protein could significantly reduce the viral load level of NNV-infected cells, indicating that fish nerve necrosis virus blocking protein can effectively block NNV infection in the striped moon goby cell line (SSN-1) cells, and the effect of blocking viral infection is concentration-dependent.

[0074] Case 2:

[0075] Feed premix additive mixing operation method: Take 5g of the feed premix additive prepared in Example 2, add it to about 30ml of water, add it while stirring to make it fully mixed until a suspension is completely formed, mix it thoroughly with 1kg of feed, and air dry it for more than 10 minutes to obtain fish feed uniformly coated with feed premix additive.

[0076] Preparation method of fish nerve necrosis virus blocking protein aluminum glue injection: The purified fish nerve necrosis virus blocking protein solution was filtered through a 0.22μm syringe filter for sterilization. The protein concentration was determined by the BCA method. The fish nerve necrosis virus blocking protein solution with a final concentration of 100μg / ml was prepared with 0.01M PBS, wherein the content of aluminum hydroxide glue adjuvant was 10% (v / v). It was stored at 5℃ and frozen.

[0077] 720 uniformly sized mandarin fish from the same batch, measuring 12-15 cm in length, were collected. All experimental fish were first challenged with nerve necrosis virus (NSV) tissue sample. 0.1 g of visceral tissue from diseased mandarin fish (NSV-positive only, CT value 10.98) was thoroughly ground, resuspended in PBS, centrifuged at 10,000 rpm, 4°C for 5 minutes, and the supernatant was filtered through a 0.22 μm syringe filter for sterilization. The solution was then diluted to 100 ml with PBS and administered intramuscularly via dorsal fin injection at a dose of 100 μL / fish. All experimental fish were then divided into four groups of 180 fish each, with three replicates of 60 fish per replicate. Each replicate was weighed and housed in identical 200L circular culture tanks equipped with inlet / outlet pipes and an aeration pump. All groups were kept under identical rearing conditions. Group 1 served as the infection control group, fed a blank diet daily. Group 2 was the half-dose oral administration group, fed fish feed coated with half the dose of fish nerve necrosis virus blocking protein feed premix additive for 7 consecutive days, followed by a normal diet. Group 3 was the oral administration group, fed fish feed coated with the normal dose of fish nerve necrosis virus blocking protein feed premix additive for 7 consecutive days, followed by a normal diet. Group 4 was the injection control group, each fish was injected intraperitoneally with 0.1 ml of fish nerve necrosis virus blocking protein aluminum gel injection preparation, and fed a normal diet daily. The daily feed intake for each group was calculated as 4% of body weight, divided into two feedings (morning and evening), with the same amount of feed each time. During the experiment, the feed intake was adjusted daily based on the decrease in feed intake and mortality of the fish in each group. The experiment was observed for 14 days, and the number of surviving fish in each group was recorded. The average mortality rate of the experimental fish in each group was calculated, and the relative protection rate of each experimental group was calculated using the formula: (Control group mortality rate - Experimental group mortality rate) / Control group mortality rate × 100%.

[0078] Fourteen days later, five tails were sampled from each parallel group of each experimental group for testing for nerve necrosis virus. The experimental results are shown in Tables 2 and 3.

[0079] Table 2 Statistical analysis of results for each group of experimental fish

[0080]

[0081] Table 3 Virus Detection Results

[0082]

[0083] According to Tables 2 and 3, in Group 1 (infection control group), the experimental fish fed with ordinary feed had an average mortality rate of 89.4% across three parallel groups. Affected fish exhibited decreased appetite or anorexia, abnormal eye and body coloration, and swam in a spiral or rotating manner, or remained belly-up when at rest. In Group 2 (half-dose oral administration test group), the fish were fed a half-dose of feed premix containing nerve necrosis virus blocking protein additive. The average mortality rate across three parallel groups was 45.0%, with a relative protection rate of 49.7%. In Group 3 (oral administration test group), the fish were fed a normal dose of feed premix containing nerve necrosis virus blocking protein additive. The average mortality rate across three parallel groups was 26.1%, with a relative protection rate of 70.8%. In Group 4 (injection control group), the fish were injected with a fish nerve necrosis virus blocking protein aluminum gel injection. The average mortality rate across three parallel groups was 22.3%, with a relative protection rate of 75.1%. After the experiment, healthy experimental fish exhibited normal swimming, feeding, body condition, and eye movements. In Group 1 (infected control group), the surviving fish tested positive for nerve necrosis virus (NSN), indicating continued NNN infection. In the other three experimental groups, the surviving fish tested negative for NNN. These results suggest that NNN blocking proteins help fish resist NNN infection. Using feed premix additives containing NNN blocking proteins can effectively block NNN infection and improve the survival rate of NNN-infected fish.

[0084] Case 3:

[0085] A grouper fry breeding and rearing base in Zhanjiang, an area with a high incidence of neuronecrosis virus disease, selected 30,000 pearl grouper fry (approximately 3 cm in length) and divided them into two groups: an experimental group and a control group, with three replicates in each group. Each group was housed in six 3m × 4m cement ponds, with 5,000 fry in each pond. Each pond had an independent water circulation system. The experimental group fish in the three ponds were fed daily with feed containing a neuronecrosis virus blocking protein premix additive, while the control group fish in the three ponds were fed regular feed. The daily feed amount for the experimental fish in each pond was calculated as 5% of their body weight, divided into three equal feedings. During the experiment, the feed amount was periodically increased based on the growth and feeding behavior of the fry. The experiment was observed for 30 days, and the survival rate of each experimental pond was recorded at the end of the experiment. The results are shown in Table 4.

[0086] Table 4 Statistical Analysis of Experimental Results

[0087]

[0088] Table 4 shows that the survival rates of experimental group 1 were 73.12%, experimental group 2 was 76.62%, and experimental group 3 was 73.96%; the survival rates of control group 1 were 32.08%, control group 2 was 34.24%, and control group 3 was 33.50%. This indicates that feeding premixed feed containing neuronecrosis virus blocking protein additives can significantly improve the survival rate of pearl grouper fry in environments with a high incidence of neuronecrosis virus disease. The neuronecrosis virus blocking protein can function in the grouper's body, exhibiting good control effects against neuronecrosis virus disease and effectively reducing the risk of death in grouper fry due to neuronecrosis virus infection.

Claims

1. A fish nerve necrosis virus blocking protein, characterized in that: The amino acid sequence of the fish nerve necrosis virus blocking protein is shown in SEQ ID NO.

1.

2. A gene encoding the fish nerve necrosis virus blocking protein of claim 1, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. A method for preparing the fish nerve necrosis virus blocking protein according to claim 1, characterized in that: The coding gene was ligated into the vector Pet-30a+ using BamHI and HindIII as restriction sites to obtain a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli BL21-DE3, and the expression of the target protein was induced by IPTG. After purification, the fish nerve necrosis virus blocking protein was obtained.

4. A feed premix additive for preventing fish nerve necrosis virus, characterized in that: The product comprises, by weight, 1-2 parts of the fish nerve necrosis virus blocking protein as described in claim 1, 350-360 parts of basic excipients, and 50-60 parts of plant extract excipients.

5. The feed premix additive for preventing fish nerve necrosis virus as described in claim 4, characterized in that: The product comprises, by weight, 1 part of the fish nerve necrosis virus blocking protein as described in claim 1, 350 parts of basic excipients, and 50 parts of plant extract excipients.

6. The feed premix additive for preventing fish nerve necrosis virus as described in claim 4, characterized in that: The basic auxiliary materials include the following raw materials in parts by weight: 2-5 parts calcium-based bentonite, 2-4 parts modified zeolite powder, 0.3-0.8 parts potassium silicate, 3-4 parts onion powder, 10-20 parts soybean flour, 4-8 parts yeast powder, 8-25 parts rice bran, 3-6 parts fish oil, 8-25 parts egg white powder, and 8-16 parts soybean meal.

7. The feed premix additive for preventing fish nerve necrosis virus as described in claim 4, characterized in that: The plant extract excipients include the following raw materials in parts by weight: Astragalus membranaceus powder 0.5-1 part, Clematis armandii 0.5-1 part, Houttuynia cordata 0.5-1 part, Viola yedoensis 0.5-1 part, Isatis indigotica 0.5-1 part, Chrysanthemum indicum 0.5-1 part, Artemisia argyi 0.1-0.8 part, Fennel 0.1-0.8 part, and Platycodon grandiflorus 0.8-2.5 parts.

8. The method for preparing the feed premix additive for preventing fish nerve necrosis virus as described in claim 4, characterized in that: Fish nerve necrosis virus blocking protein, basic excipients and plant extract excipients are added to a mixer in sequence and mixed thoroughly to obtain a feed premix additive for preventing fish nerve necrosis virus.

9. The use of the fish nerve necrosis virus blocking protein as described in claim 1 or the feed premix additive for preventing fish nerve necrosis virus as described in claim 4 in the preparation of feed for preventing fish nerve necrosis virus.

10. The application as described in claim 9, characterized in that: The feed premix additive for preventing fish nerve necrosis virus is added to the feed for preventing fish nerve necrosis virus at a mass percentage of 0.1%-1%.

Images