Fish nervous necrosis virus blocking protein as well as preparation method and application thereof

Abstract

The invention 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 of the protein, the protein can be specifically bonded with receptors on the surfaces of fish nervous necrosis virus particles, so that the fish nervous necrosis virus particles lose the infection capability on fish somatic cells, the fish nervous necrosis virus is prevented from infecting fish host cells, and the fish nervous necrosis virus is prevented from being copied and proliferated in a host. The fish nervous necrosis virus blocking protein provided by the invention has an efficient nervous necrosis virus resisting effect, a biological preparation product prepared by taking the fish nervous necrosis virus blocking protein as an active material can remarkably improve the nervous necrosis virus resisting capability of fry in a mixing oral administration manner, is safe and pollution-free, can be subjected to large-scale industrial application, and has a wide application prospect. A new way is provided for preventing and controlling the nervous necrosis virus disease of the fish, so that the economic benefit of fish culture is improved.

Description

Technical Field

[0001] The present invention belongs to the field of prevention and control of aquatic pathogenic microorganisms, and particularly relates to a fish nervous necrosis virus blocking protein, a preparation method thereof, and an application thereof. Background Art

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

[0003] Currently, the prevention and control measures for fish nervous necrosis virus disease are very limited. Although vaccination is a relatively common way to prevent the virus infection, the vaccine for fish nervous necrosis virus disease is still in the primary research and development stage, and the role it can play for fry with imperfectly developed immune systems is relatively limited. Therefore, developing safe and efficient anti-fish nervous necrosis virus antiviral preparations has important practical significance. Summary of the Invention

[0004] Aiming at the problems in the prior art, the present invention provides a fish nervous necrosis virus blocking protein. Due to its special structure, this protein can specifically bind to the receptor on the surface of fish nervous necrosis virus particles, making it lose the ability to infect fish somatic cells, thereby blocking the infection of fish nervous necrosis virus to fish host cells and preventing it from replicating and proliferating in the host body. The fish nervous necrosis virus blocking protein provided by the present invention has a highly efficient anti-nervous necrosis virus effect. The biological preparation product prepared with this as the active material can significantly improve the anti-nervous necrosis virus ability of fry when used by mixing with feed and orally, and is safe and pollution-free, and can be applied on a large scale industrially, providing a new way for the prevention and control of fish nervous necrosis virus disease, and further enhancing the economic benefits of fish farming.

[0005] The object of the present invention can be achieved by the following technical solutions: In the first aspect of the present invention, a fish nervous necrosis virus blocking protein is provided, characterized in that: the amino acid sequence of the fish nervous necrosis virus blocking protein is as shown in SEQ ID NO.1.

[0006] In the second aspect of the present invention, a gene encoding the above-mentioned fish nervous necrosis virus blocking protein is provided, and the nucleotide sequence of the gene is as shown in SEQ ID NO.2.

[0007] The third aspect of the present invention provides a method for preparing the above-mentioned fish nervous necrosis virus blocking protein, including: ligating the coding gene with Bam H I and Hin d Ⅲ as restriction enzyme sites to the vector Pet-30a(+), obtaining a recombinant plasmid; transforming the recombinant plasmid into Escherichia coli BL21-DE3, inducing the expression of the target protein by IPTG, and obtaining the fish nervous necrosis virus blocking protein after purification.

[0008] The fourth aspect of the present invention provides a feed premix additive for preventing fish nervous necrosis virus. By mass fraction, it includes 1-2 parts of the above-mentioned fish nervous necrosis virus blocking protein, 350-360 parts of basic excipients, and 50-60 parts of plant extract excipients.

[0009] Preferably, by mass fraction, it includes 1 part of the above-mentioned fish nervous necrosis virus blocking protein, 350 parts of basic excipients, and 50 parts of plant extract excipients.

[0010] Furthermore, the basic excipients include raw materials with the following mass fractions: 2-5 parts of calcium-based bentonite, 2-4 parts of modified zeolite powder, 0.3-0.8 parts of potassium silicate, 3-4 parts of onion powder, 10-20 parts of soybean powder, 4-8 parts of yeast powder, 8-25 parts of rice bran, 3-6 parts of fish oil, 8-25 parts of egg white powder, and 8-16 parts of soybean meal.

[0011] Among them, the particle fineness of the basic excipients is above 100 mesh.

[0012] Calcium-based bentonite is used as a mineral supplement.

[0013] Modified zeolite powder is used as an adsorbent to improve the intestinal environment.

[0014] Potassium silicate is used as a dispersant to promote the mutual dispersion of each raw material.

[0015] Onion powder has antibacterial and immune-enhancing effects.

[0016] Soybean powder and yeast powder are used as sources of protein and B vitamins.

[0017] Rice bran is used as a source of fiber.

[0018] Fish oil is used as an energy source.

[0019] Egg white powder and soybean meal are used as sources of protein.

[0020] Furthermore, the plant extract auxiliary materials include raw materials in the following parts by mass: 0.5 - 1 part of astragalus powder, 0.5 - 1 part of grand blueberry leaf, 0.5 - 1 part of houttuynia cordata, 0.5 - 1 part of viola philippica, 0.5 - 1 part of isatis root, 0.5 - 1 part of wild chrysanthemum, 0.1 - 0.8 part of mugwort leaf, 0.1 - 0.8 part of fennel, and 0.8 - 2.5 parts of platycodon grandiflorum.

[0021] Among them, the particle fineness of the plant extract auxiliary materials is above 100 mesh.

[0022] Astragalus, grand blueberry leaf, houttuynia cordata, viola philippica, isatis root, and wild chrysanthemum are used as natural antiviral and immune enhancers.

[0023] Mugwort leaf and fennel have a flavoring effect and are beneficial to the health of the body.

[0024] Platycodon grandiflorum has an immunomodulatory effect.

[0025] The fifth aspect of the present invention provides a preparation method of a feed premix additive for preventing fish nervous necrosis virus as described above: adding the fish nervous necrosis virus blocking protein, basic auxiliary materials, and plant extract auxiliary materials as described above to a stirrer in sequence, and fully stirring and mixing evenly by the stirrer to obtain a feed premix additive for preventing fish nervous necrosis virus.

[0026] The sixth aspect of the present invention provides an application of the fish nervous necrosis virus blocking protein as described above or the feed premix additive for preventing fish nervous necrosis virus as described above in the preparation of a feed for preventing fish nervous necrosis virus.

[0027] Furthermore, the feed premix additive for preventing fish nervous necrosis virus is added to the feed for preventing fish nervous necrosis virus in a mass percentage of 0.1% - 1%.

[0028] The beneficial effects obtained by one or more of the above technical solutions of the present invention are as follows: The fish nervous necrosis virus blocking protein provided by the present invention can specifically bind to the receptor on the surface of fish nervous necrosis virus particles, making it lose the ability to infect fish somatic cells, thereby blocking the infection of nervous necrosis virus to fish host cells, preventing its replication and proliferation in the host, and achieving an efficient anti - nervous necrosis virus effect. When this protein is added to the feed premix additive, it can significantly improve the anti - nervous necrosis virus ability of the body, and is safe and pollution - free, and can be applied on a large scale industrially, thereby enhancing the economic benefits of fish farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a schematic diagram of the induced expression result of the target protein of the fish nervous necrosis virus blocking protein of the present invention; Figure 2Schematic diagram of the SDS-PAGE result after purification of the fish nervous necrosis virus blocking protein of the present invention. Detailed implementation manners

[0030] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments.

[0031] The materials, reagents and experimental equipment involved in the embodiments of the present invention are all commercially available products unless otherwise specified.

[0032] Embodiment 1 A fish nervous necrosis virus blocking protein, its amino acid sequence (SEQ ID NO.1): GGGGSGSRRFPPLPAVIEEPMETDGGGGSGGGGSGGGGSMVRKGEKKLAKPGGGGSGTTKAANPQPRRRGGGGSGANNRRRSNRTDAPVSGGGGSGLPDPTDNDHTFGGGGSGWESRTVRPQYTRGGGGSGSSGKEQRLTSPGRGGGGSGLETPEETTAGGGGSGDSLSTNDFKSGGGGSGLGTGDVDRAVYWSGGGGSGkkkkWDNFNKTFTDSGGGGSGYYSDEQPRQILLGGGGSGVGTVCTRVDSENGGGGSGKKKKNNRRRSNRTDAPGGGGSGIEGR Through the gene sequence (SEQ ID NO.2) optimized for Escherichia coli expression codons: GGTGGTGGTGGTTCTGGTTCTCGTCGTTTCCCGCCGCTGCCGGCTGTTATCGAAGAACCGATGGAAACCGACGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTATGGTTCGTAAAGGTGAAAAAAAACTGGCTAAACCGGGTGGTGGTGGTTCTGGTACCACCAAAGCTGCTAACCCGCAGCCGCGTCGTCGTGGTGGTGGTGGTTCTGGTGCTAACAACCGTCGTCGTTCTAACCGTACCGACGCTCCGGTTTCTGGTGGTGGTGGTTCTGGTCTGCCGGACCCGACCGACAACGACCACACCTTCGGTGGTGGTGGTTCTGGTTGGGAATCTCGTACCGTTCGTCCGCAGTACACCCGTGGTGGTGGTGGTTCTGGTTCTTCTGGTAAAGAACAGCGTCTGACCTCTCCGGGTCGTGGTGGTGGTGGTTCTGGTCTGGAAACCCCGGAAGAAACCACCGCTGGTGGTGGTGGTTCTGGTGACTCTCTGTCTACCAACGACTTCAAATCTGGTGGTGGTGGTTCTGGTCTGGGTACCGGTGACGTTGACCGTGCTGTTTACTGGTCTGGTGGTGGTGGTTCTGGTAAAAAAAAAAAATGGGACAACTTCAACAAAACCTTCACCGACTCTGGTGGTGGTGGTTCTGGTTACTACTCTGACGAACAGCCGCGTCAGATCCTGCTGGGTGGTGGTGGTTCTGGTGTTGGTACCGTTTGCACCCGTGTTGACTCTGAAAACGGTGGTGGTGGTTCTGGTAAAAAAAAAAAAAACAACCGTCGTCGTTCTAACCGTACCGACGCTCCGGGTGGTGGTGGTTCTGGTATCGAAGGTCGT The preparation method of this fish nervous necrosis virus blocking protein is as follows: Based on the above gene sequence, the restriction enzyme cleavage sites BamHI and HindⅢ were introduced, synthesized by Shanghai Sangon Biotech Co., Ltd., and cloned and ligated to the corresponding cleavage sites of the vector pET-30a(+) through the restriction enzyme cleavage sites at both ends of the gene sequence. It was transformed into the Escherichia coli DH5α cloning strain by the heat shock method, spread on the LB solid medium containing 50 μg / ml Kan+, grown, the transformants containing the recombinant plasmid were screened out by colony PCR method, and inoculated into the LB liquid medium containing 50 μg / ml Kan+ and cultured at 37 °C with shaking at 200 rpm for 12 hours, and the recombinant plasmid was extracted.

[0033] The obtained recombinant plasmid containing the target gene fragment was transformed into Escherichia coli BL21(DE3) competent cells by the heat shock method, and the transformed cells were spread on the LB solid medium containing 50 μg / ml Kan+ and grown, and the BL21(DE3) transformants containing the recombinant plasmid were screened out.

[0034] The single colony of BL21(DE3) bacteria containing the recombinant plasmid grown on the Kan+ resistant plate was picked with a sterilized toothpick and inoculated into 10 ml of LB liquid medium containing 50 μg / ml Amp+ and cultured overnight at 37 °C with shaking at 200 rpm in a constant temperature shaker. The next day, the bacterial liquid was transferred to the LB liquid medium containing 50 μg / ml Amp+ at a ratio of 1:100, and when the OD600 value reached 0.5 (the time was 3 hours), the IPTG stock solution (100 mmol / L) was added at a ratio of 1:200, and the culture was continued for 5 h to induce the expression of the target protein. The verification of the induced expression of the target protein of the fish nervous necrosis virus blocking protein was as Figure 1 shown (M: 180 kDa colored pre-stained protein molecular weight standard; lane 1: bacterial liquid without IPTG induction; lanes 2-3: bacterial liquid induced by IPTG).

[0035] Purification of fish nervous necrosis virus blocking protein: The E. coli fermentation broth expressing the recombinant protein was centrifuged at 5000rpm for 8 minutes to collect the cells, and the cells were resuspended in PBS with 1 / 20 volume of the fermentation broth, and the cells were homogenized and broken by a high-pressure homogenizer, and protease inhibitors were added during the breaking process. The broken liquid mixture was centrifuged again at 13000rpm, 4℃, 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 the precipitate was collected by centrifugation at 10000rpm after oscillation at room temperature for 20 minutes. This was repeated twice to remove most of the soluble impurities. The washed precipitated protein was dissolved in a PBS solution containing 8M urea, oscillated at room temperature for 20 minutes until the precipitated protein was fully dissolved and free of white particles, and centrifuged at 13000rpm at 4℃ to remove cell debris and unbroken cells, and the supernatant was collected to obtain the purified protein. The SDS-PAGE verification chart of the purified fish neuronecrosis virus blocking protein is as follows Figure 2 As shown (M: 180 kDa color pre-stained protein molecular weight standard; lane 1: protein solution before purification; lane 2: protein washing solution; lane 3: purified protein solution of collection 1; lane 4: purified protein solution of collection tube 2; lane 5: purified protein solution of collection tube 3), the purified protein obtained was spray-dried and stored at 5°C for future use.

[0036] Example 2 This embodiment provides a feed premix additive for preventing fish neuronecrosis virus: Basic auxiliary materials (particle fineness is 100 mesh): 3 parts of calcium-based bentonite, 2 parts of modified zeolite powder, 0.5 parts of potassium silicate, 3 parts of onion powder, 10 parts of soybean powder, 4 parts of yeast powder, 10 parts of rice bran, 5 parts of fish oil, 10 parts of egg white powder, and 12 parts of soybean meal.

[0037] Plant extract excipients (particle size is 100 mesh): 0.5 parts of astragalus powder, 0.5 parts of large-leaf green, 0.5 parts of houttuynia, 0.5 parts of viola yedoensis, 0.5 parts of isatis root, 0.5 parts of wild chrysanthemum, 0.3 parts of wormwood, 0.3 parts of fennel, and 2 parts of platycodon.

[0038] One part of fish nervous necrosis virus blocking protein, 350 parts of basic auxiliary materials and 50 parts of plant extract auxiliary materials are sequentially added into a blender, and the mixture is fully stirred by the blender to obtain a feed premix additive for fish nervous necrosis virus.

[0039] Example 3 Performance evaluation of the effect of blocking fish neuronecrosis virus: Case 1: To verify the ability of fish neural necrosis virus blocking protein to block neural necrosis virus (NNV) infection at the cellular level.

[0040] Experimental Materials Cell line: Cells of the striped snakehead cell line (SSN-1); Virus: Nervous necrosis virus (NNV); Blocking protein: Purified nervous necrosis virus blocking protein (NNV-BP), sterilized by filtration through a 0.22 μm syringe filter; Culture medium: Leibovitz's L-15 medium; Other reagents: PBS buffer, trypsin, fetal bovine serum (FBS), antibiotics (such as penicillin-streptomycin).

[0041] Experimental procedures A. SSN-1 cells cultured in L-15 medium containing 10% FBS and 1% antibiotics at 37 °C and 5% CO2 were digested with trypsin and passaged when the cell monolayer was confluent in the cell flask.

[0042] B. The cells were seeded into a 12-well cell culture plate at 2×10 5 cells per well and cultured for about 48 hours. The infection experiment was carried out when more than 90% of the cells adhered to the wall.

[0043] C. The fish nervous necrosis virus blocking protein was diluted to different concentrations (0 μg / ml, 0.1 μg / ml, 1 μg / ml, 10 μg / ml) with serum-free L-15 medium. They were labeled as the infection control group, low-concentration experimental group, medium-concentration experimental group, and high-concentration experimental group in turn.

[0044] D. Different concentrations of the fish nervous necrosis virus blocking protein were added to the well-cultured SSN-1 cells in the 12-well cell culture plate, with 3 parallel wells for each concentration, and incubated at 37 °C for 1 hour.

[0045] E. The NNV cell culture virus solution was centrifuged to remove cell debris, and the virus solution was diluted with serum-free L-15 medium.

[0046] F. After the incubation of the nervous necrosis virus blocking protein was completed, the culture medium was aspirated, and the medium containing NNV (MOI = 1) was added, and incubated at 37 °C for 1 hour. The virus solution was aspirated, and the cells were washed 3 times with PBS, standing for 3 - 5 minutes each time, and then fresh complete medium was added for continued culture.

[0047] G. Cells were collected 24 hours after infection. Another group of uninfected cells was used as the blank control group. Total RNA of each group of cell samples was extracted, reverse transcribed into cDNA, and the virus load levels of each group of samples were detected by qPCR.

[0048] The results are shown in Table 1: Table 1 Virus load of cells infected with nervous necrosis virus treated with different concentrations of nervous necrosis virus blocking protein

[0049] As can be seen from Table 1, the CT value of the infected control group cells not treated with the nerve necrosis virus blocking protein for the detection of the nerve necrosis virus was 21.03, indicating that the virus was successfully infected and replicated. The CT value of the nerve necrosis virus detection in the low-concentration experimental group cells was 28.52, the CT value of the medium-concentration experimental group cells was 32.45, there was no CT value for the high-concentration experimental group cells, and there was no CT value for the blank cell control, serving as a valid negative control for the experiment. It can be seen that as the concentration of the nerve necrosis virus blocking protein increases, the CT value of the nerve necrosis virus detection increases and the viral load decreases. The high-concentration blocking protein may effectively block the infection of the nerve necrosis virus.

[0050] The experimental results show that the fish nerve necrosis virus blocking protein can significantly reduce the viral load level of NNV-infected cells, indicating that the fish nerve necrosis virus blocking protein can effectively block the infection of NNV in cells of the striped snakehead cell line (SSN-1), and the effect of blocking virus infection is concentration-dependent.

[0051] Case 2: Method for mixing the feed premix additive with feed: Take 5 g of the feed premix additive prepared in Example 2, add it to about 30 ml of water, add it while stirring to make it fully mixed until a suspension is completely formed, stir it well with 1 kg of feed, and air-dry it for more than 10 minutes to obtain fish feed uniformly coated with the feed premix additive.

[0052] Preparation method of the aluminum adjuvant injection preparation of the fish nerve necrosis virus blocking protein: The purified fish nerve necrosis virus blocking protein solution is filtered and sterilized with a 0.22 μm needle filter, its protein concentration is determined by the BCA method, and a fish nerve necrosis virus blocking protein solution with a final concentration of 100 μg / ml is prepared with 0.01 M PBS, wherein the content of the aluminum hydroxide adjuvant is 10% (v / v), and it is stored at 5°C to avoid freezing.

[0053] Take 720 feed mandarin fish of the same batch with uniform size, 12 - 15 cm in body length. First, all experimental fish were challenged with the tissue poison of nervous necrosis virus. Take 0.1 g of visceral tissue of diseased mandarin fish infected with nervous necrosis virus (only positive for nervous necrosis virus detection, CT value is 10.98), grind it thoroughly, resuspend it with an appropriate amount of PBS, centrifuge at 10000 rpm for 5 minutes at 4 °C, take the supernatant and filter it through a 0.22 μm needle filter to sterilize, and then make up the volume to 100 ml with PBS as the virus solution. Inject it into the dorsal fin muscle, and the challenge dose is 100 μL per fish. Then divide all the experimental fish into 4 groups evenly, with 180 fish in each group. Set 3 parallel groups in each group, with 60 fish in each parallel group. After weighing them respectively, raise them in 200 L circular culture barrels of the same size, configure the inlet and outlet pipes and aeration pumps, and keep the feeding conditions of the experimental fish in each group consistent. The first group is the infection control group, which is fed with blank fish feed every day. The second group is the half-dose oral test group, which is fed with fish feed wrapped with half-dose of the premix additive of nervous necrosis virus blocking protein for 7 consecutive days, and then fed with ordinary feed. The third group is the oral test group, which is fed with fish feed wrapped with normal-dose of the premix additive of nervous necrosis virus blocking protein for 7 consecutive days, and then fed with ordinary feed. The fourth group is the injection control group, and each fish is intraperitoneally injected with 0.1 ml of the aluminum adjuvant injection preparation of nervous necrosis virus blocking protein, and fed with ordinary feed every day. The daily feed intake of the experimental fish in each group is calculated according to 4% of the body weight, and is fed twice (in the morning and evening), and the feed intake each time is the same. During the experiment, the feed intake is calculated and adjusted every day according to the decrease in feed intake and the death situation of the experimental fish in each group due to disease. Observe the experiment for 14 days and record the survival number of the experimental fish in each group. Calculate the average mortality rate of the experimental fish in each test group, and calculate the relative protection rate of each test group according to the formula relative protection rate = (control group mortality rate - test group mortality rate) / control group mortality rate × 100%.

[0054] After 14 days, 5 fish from each parallel group of each test group were sampled and mixed for nervous necrosis virus detection, and the experimental results are shown in Table 2 and Table 3.

[0055] Table 2 Statistical results of experimental fish in each group

[0056] Table 3 Virus detection results

[0057] According to Tables 2 and 3, for the experimental fish in Group 1 (infected control group) fed with ordinary feed, the average mortality rate of the 3 parallel groups was 89.4%. Moreover, the diseased fish had a decreased appetite or anorexia, abnormal eyes and body color, swam in a spiral or rotary manner, or had their bellies facing upwards when stationary. In Group 2 (half-dose oral test group), the fish were fed with feed wrapped with a half-dose of feed premix additive containing a nerve necrosis virus blocking protein, and the average mortality rate of the 3 parallel groups was 45.0%, with a relative protection rate of 49.7%. In Group 3 (oral test group), the fish were fed with feed wrapped with a normal dose of feed premix additive containing a nerve necrosis virus blocking protein, and the average mortality rate of the 3 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 adjuvant injection preparation, and the average mortality rate of the 3 parallel groups was 22.3%, with a relative protection rate of 75.1%. After the experiment, the surviving healthy experimental fish swam, fed, and had normal bodies and eyes. The surviving fish in Group 1 (infected control group) were detected positive for the nerve necrosis virus after the experiment, and there was still a nerve necrosis virus infection. The surviving fish in the other 3 test groups were all detected negative for the nerve necrosis virus after the experiment. The results showed that the fish nerve necrosis virus blocking protein helped the fish body resist the infection of the nerve necrosis virus, and using a feed premix additive containing the fish nerve necrosis virus blocking protein could effectively block the infection of the nerve necrosis virus and improve the survival rate of fish infected with the nerve necrosis virus.

[0058] Case 3: A certain grouper fry cultivation and breeding base with a high incidence of nerve necrosis virus disease in Zhanjiang was selected. 30,000 pearl gentian grouper fry with a body length of about 3 cm were taken and evenly divided into two groups, namely the experimental group and the control group, with 3 parallels in each group. They were respectively raised in 6 cement ponds of 3m×4m, with 5000 fish in each pond, and each pond was equipped with an independent water circulation system. The experimental fish in the three ponds of the experimental group were fed with feed wrapped with a feed premix additive containing a nerve necrosis virus blocking protein every day, and the fish in the 3 ponds of the control group were fed with ordinary feed every day. The daily feed feeding amount of the test fish in each pond was calculated according to 5% of the body weight and was fed in 3 times, with the same amount of feed fed each time. During the experiment, according to the growth and feeding situation of the fry, the feed feeding amount was regularly increased. The experiment was observed for 30 days, and the survival rate of each experimental pond was counted after the experiment. The experimental results are shown in Table 4.

[0059] Table 4 Statistical Results of the Experiment

[0060] As can be seen from Table 4, the survival rate of experimental group 1 was 73.12%, that of experimental group 2 was 76.62%, and that of experimental group 3 was 73.96%; the survival rate of control group 1 was 32.08%, that of control group 2 was 34.24%, and that of control group 3 was 33.50%. This shows that feeding the feed with the premix additive of the nerve necrosis virus blocking protein can significantly improve the survival rate of pearl gentian grouper fry in the environment with a high incidence of nerve necrosis virus disease. The nerve necrosis virus blocking protein can play a role in the grouper body, has a good prevention and control effect on nerve necrosis virus disease, and can effectively reduce the death risk of grouper fry caused by infection with nerve necrosis virus.

Claims

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

1.

2. A gene encoding the fish nervous necrosis virus blocking protein according to 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 nervous necrosis virus blocking protein according to claim 1, characterized in that: The coding gene was connected to 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, the expression of the target protein was induced by IPTG, and the fish neuronecrosis virus blocking protein was obtained after purification.

4. A feed premix additive for preventing fish nervous necrosis virus, characterized in that: Calculated by weight, it comprises 1-2 parts of the fish neuronecrosis virus blocking protein according to claim 1, 350-360 parts of basic auxiliary materials and 50-60 parts of plant extract auxiliary materials.

5. The feed premix additive for preventing fish nervous necrosis virus according to claim 4, wherein: Calculated by weight, it comprises 1 part of the fish neuronecrosis virus blocking protein according to claim 1, 350 parts of basic auxiliary materials and 50 parts of plant extract auxiliary materials.

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

7. The feed premix additive for preventing fish neural necrosis virus according to claim 4, characterized in that: The plant extract auxiliary materials include the following raw materials in parts by weight: 0.5-1 part of astragalus powder, 0.5-1 part of large-leaf green, 0.5-1 part of houttuynia cordata, 0.5-1 part of viola yedoensis, 0.5-1 part of isatis root, 0.5-1 part of wild chrysanthemum, 0.1-0.8 part of wormwood, 0.1-0.8 part of fennel, and 0.8-2.5 parts of platycodon.

8. The preparation method of the feed premix additive for preventing fish nervous necrosis virus according to claim 4, characterized in that: The fish neuronecrosis virus blocking protein, basic auxiliary materials and plant extract auxiliary materials are sequentially added into a stirrer, and the mixture is fully and evenly stirred by the stirrer to obtain a feed premix additive for preventing fish neuronecrosis virus.

9. Use of the fish nervous necrosis virus blocking protein according to claim 1 or the feed premix additive for preventing fish nervous necrosis virus according to claim 4 in preparing feed for preventing fish nervous necrosis virus.

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

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