Egg yolk antibody for nervous necrosis virus of grouper as well as preparation method and application of egg yolk antibody

Abstract

The invention provides a preparation method of a grouper nervous necrosis virus protein antigen and an egg yolk antibody, which comprises the following steps of: obtaining a grouper nervous necrosis virus capsid protein gene sequence, recombining the grouper nervous necrosis virus capsid protein gene sequence into a pET-28a carrier, and introducing into escherichia coli for expression to obtain the grouper nervous necrosis virus protein antigen. The grouper nervous necrosis virus capsid protein antigen obtained after purification, concentration, folding and dialysis is pseudovirus with an icosahedron symmetrical structure, after laying hens are immunized by the pseudovirus, eggs of the laying hens are collected, and egg yolk antibodies are extracted and applied to immune protection of groupers. The obtained antigen is high in immunogenicity, the obtained antibody has excellent titer, has practical value for preventing and treating nervous necrosis virus of grouper, is high in safety and low in cost, and can effectively reduce economic loss of aquaculture.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and virus prevention and control, and particularly relates to a high-titer yolk antibody against nervous necrosis virus of grouper, a preparation method thereof, and an application thereof. Background Art

[0002] Grouper is a marine animal with important economic value. Compared with some other seawater fish species, grouper has the advantage of a fast growth rate, can reach the market specification relatively quickly, and has a short breeding cycle. Due to its good taste (known as "sea chicken") and rich nutritional value, the demand for grouper in domestic and foreign markets is relatively large. With the gradual maturity of artificial breeding technology, its breeding scale has been continuously expanding. Nervous necrosis virus (NNV) is the most seriously harmful virus in grouper breeding. Once it breaks out when there is no effective drug control at present, it will cause extremely heavy economic losses. Nervous necrosis virus belongs to the genus Betanodavirus of the family Nodaviridae. The virus morphology is an icosahedron without an envelope, with a size of about 25 nm. Its RNA genome consists of two single-stranded positive-sense RNAs, RNA1 and RNA2, which encode RNA polymerase (RdRp) and capsid protein (CP) respectively. NNV is widely distributed globally, especially in aquaculture farms in Asia, Europe, Australia, Africa, and the Americas. It can affect the larvae, juveniles, and even adults of more than 30 important economic fish species. The positive rates of NNV detected by spot checks in 2018 - 2020 were 29%, 18.2%, and 12.3% respectively, which is a virus with a relatively high detection rate among farmed fish viruses. Viral nervous necrosis in fish, also known as viral encephalomyelonecrosis in fish, was initially discovered in fish farms in Japan and Europe in the late 1980s. Subsequently, research confirmed that this is a new virus belonging to the family Nodaviridae. This virus mainly affects the central nervous system of fish, causing vacuolar necrosis in the brain and retina, manifested as abnormal behavior, difficulty in swimming, loss of appetite, etc., and the mortality rate can reach up to 100%. At present, there is a lack of effective means for the treatment of grouper nervous necrosis virus. Key measures for the prevention and control of NNV infection include strengthening the sanitation management of the aquaculture environment, using healthy seedlings, regular detection and monitoring, etc. For infected fish populations, isolation and treatment measures need to be taken to reduce virus transmission and losses. At present, the NNV vaccine has a certain prevention and control effect on the virus, but there are problems such as weak immune effect, insufficient immunogenicity, and potential integration risks. At the same time, its application effect is limited by various factors such as the health status of fish and the aquaculture environment. Egg yolk antibody is an immunoglobulin extracted from egg yolk, also known as IgY. Its molecular weight is relatively large, which can better recognize and bind to macromolecular antigens. In addition, IgY has a longer half-life in the body and can maintain immune protection for a longer time. Egg yolk antibody can be obtained from eggs without slaughtering animals, which conforms to animal rights protection, and has the characteristics of high specificity, non-toxic and side effects-free, and environmentally friendly, and is widely used in the diagnosis and prevention and treatment of animal infectious diseases. At present, some application studies of egg yolk antibody in the treatment of aquatic animals, such as Vibrio, iridovirus, cyprinid herpesvirus, etc., have been carried out by scientific research personnel. At present, there is no effective prevention and control means for the treatment of nervous necrosis virus, especially in the juvenile fish stage. Therefore, the research on the prevention and control of nervous necrosis virus by egg yolk antibody has application prospects. Summary of the Invention

[0003] Technical problems to be solved: At present, there is no specific drug for the treatment of nervous necrosis virus disease, and vaccines are mainly used as preventive means. However, vaccines have the following deficiencies: The infection of nervous necrosis virus in fish mainly occurs in the juvenile fish stage, and juvenile fish do not yet have the ability to produce antibodies. Therefore, the application value of vaccines in the juvenile fish stage is limited; Vaccines rely on injection for immunization. The fry are small in size and live in water. Injection immunization is time-consuming and inconvenient to operate, which limits the commercial promotion of vaccines, and the cost of large-scale production of fish vaccines is relatively high. While egg yolk antibody, as an antibody, is fed in the form of feed or can also be used by soaking in water. The process is simple and not affected by immune ability. The highly effective antibody prepared by the present invention not only solves the above problems, but also has a good prevention and control effect.

[0004] Technical solution: The present invention provides a grouper nervous necrosis virus capsid protein antigen, and the gene sequence of the antigen is shown in SEQ ID NO.1. Further, the grouper is Epinephelus coioides. Further, the application of the antigen in the preparation of a drug for preventing and treating nervous necrosis virus. An egg yolk antibody against grouper nervous necrosis virus, which is obtained by purifying the egg yolk of eggs laid by laying hens immunized with an antigen expressed by recombinant the gene sequence of grouper nervous necrosis virus capsid protein. Further, the application of the egg yolk antibody in the preparation of a drug for preventing and treating nervous necrosis virus. A method for preparing yolk antibody of grouper nervous necrosis virus, the steps of which are as follows: S1. Antigen protein expression: Recombine the coding gene fragment of the grouper nervous necrosis virus capsid protein shown in SEQ ID NO.1 into the pET-28a vector to construct the pET-28a-CP recombinant plasmid, and introduce it into Escherichia coli competent cells for expression; S2. Antigen protein purification: Expand the culture of Escherichia coli competent cells, and after disruption, affinity chromatography, concentration, folding dialysis, and size exclusion chromatography, obtain a high-concentration antigen protein; S3. Immunization of laying hens: Emulsify the antigen protein prepared in step S2 with an adjuvant into a vaccine, inject it into healthy hens, and collect their eggs; S4. Purification of yolk antibody: Clean and disinfect the eggs, separate the egg yolks, aspirate the egg yolk liquid, and obtain the yolk antibody after purification. Further, the process of folding dialysis in step S2 is to add buffer1 drop by drop at a flow rate of 1.5 - 2.5 mL / min with a constant flow pump in a low-temperature environment. After the addition is completed, discard half of the solution, and then add buffer2 at a flow rate of 0.5 - 1.5 mL / min. Further, the buffer1 is a mixed solution of Tris-HCl, NaCl, and (NH4)2SO4; the buffer2 is a mixed solution of Tris-HCl, NaCl, Glycerol, and CaCl2. Further, the column for size exclusion chromatography in step S2 is Superose TM 6Increase 10 / 300GL column. Further, the purification method in step S4 is the ammonium sulfate precipitation method. Further, the application of the yolk antibody of grouper nervous necrosis virus in the preparation of drugs for preventing and treating grouper viral nervous necrosis. Beneficial effects: 1. In the present invention, the gene encoding the grouper nervous necrosis virus capsid protein is recombined onto the pET-28a vector, and the antigen protein is obtained after prokaryotic expression and purification. The yield of prokaryotic culture is high, the cycle is short, and the obtained antigen protein does not contain the viral genome, cannot replicate or cause disease, and does not require inactivation or attenuation treatment; 2. Ammonium sulfate is added during the purification process of the antigen protein in the present invention to reduce electrostatic repulsion, promote the aggregation of protein molecules, and contribute to the formation of the core structure of VLP, thereby optimizing the performance. The subsequent introduction of calcium ions drives the correct assembly of the antigen protein into pseudoviruses, which have an icosahedral symmetric particle structure, mimic the viral capsid, present natural conformational epitopes, are more easily recognized by the immune system, stimulate a stronger immune response, have a stronger anti-protease degradation ability, an extended in vivo half-life, and higher immunogenicity; 3. The yolk antibody against grouper nervous necrosis virus of the present invention can be obtained from eggs without sacrificing animals, which is in line with animal rights protection. It has high specificity, no toxicity or side effects, is environmentally friendly, and the obtained yolk antibody has a high titer value, reaching 1×10 7 level, and can be maintained for a relatively long time; 4. The yolk antibody against grouper nervous necrosis virus of the present invention can be used to prevent and treat diseases caused by nervous necrosis virus, and can be used as a new feed additive to replace antibiotics, avoiding the abuse of antibiotics in the aquaculture process, which leads to drug residues in animal products, increases the resistance of bacteria to antibiotics, is more convenient to operate than injection immunization, has high application value, low cost, and is suitable for commercial promotion; 5. The present invention uses the ammonium sulfate precipitation method to purify the yolk antibody, obtaining an antibody with a high titer value. The operation is simple, without the need for sophisticated purification equipment, the cost is low, and the stability of the yolk antibody is high, which can be applied in feeds, etc.; 6. The yolk antibody of the present invention not only has the function of specific immunity, but also can improve intestinal function. The yolk antibody can inhibit the growth of harmful bacteria and promote the reproduction of beneficial bacteria, thereby improving the intestinal microecological balance. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is the SDS-PAGE electrophoresis result diagram of the antigen protein under different eluent concentrations. M, P, S, and FL respectively represent Marker, precipitate, supernatant, and flow-through, and W and E1-E3 respectively represent samples containing 0 mM, 20 mM, 50 mM, and 300 mM imidazole eluent; Figure 2 is the chromatogram of the antigen protein by size exclusion chromatography; Figure 3 is the Western blot verification diagram of the antigen protein. M represents Marker, 1 is the antigen protein after folding and dialysis treatment, and 2 represents the antigen protein without folding and dialysis; Figure 4 is the negative staining electron microscopy diagram of the antigen protein after folding and dialysis treatment; Figure 5 is the diagram of immunized laying hens and collected egg samples; Figure 6This is the SDS-PAGE electrophoresis diagram of egg yolk antibody, 1 is the egg yolk antibody obtained by chloroform extraction, 2 is the egg yolk antibody obtained by polyethylene glycol precipitation extraction, 3 is the egg yolk antibody obtained by phenol extraction, 4 is the egg yolk antibody obtained by ammonium sulfate precipitation extraction, and 5 is the egg yolk antibody obtained by water dilution-filtration method; Figure 7 It is the efficacy value graph of the egg yolk antibody detected by ELISA for grouper nervous necrosis virus; Figure 8 This is a regular diagram showing the effect of immunization time on the growth and decline of egg yolk antibodies in laying hens; Figure 9 This is a Western blot validation diagram of the specific detection of egg yolk antibodies against grouper nervous necrosis virus. M represents Marker, 1 and 2 are incubated with egg yolk antibodies from unimmunized laying hens, 3 and 4 are incubated with egg yolk antibodies, 1 and 3 substrates are antigenic proteins, and 2 and 4 substrates are other proteins similar to the antigenic proteins; Figure 10 This is a performance test chart of egg yolk antibody of grouper nervous necrosis virus, A represents the effect of temperature treatment on the activity of egg yolk antibody, B represents the effect of pH treatment on the activity of egg yolk antibody, C represents the effect of pepsin on the activity of egg yolk antibody, and D represents the effect of seawater immersion time on the activity of egg yolk antibody; Figure 11 This is a sample picture of lyophilized and reconstituted egg yolk antibody; Figure 12 This is a diagram of the breeding process of grouper; Figure 13 This is a diagram showing the protective effect of yolk antibodies on grouper; Figure 14 These are paraffin sections of grouper brain and eye tissues. A represents the blank control group, B represents the virus-infected group, and C represents the yolk antibody-immersion treated group. Figure 15 This is a BugBase species bar chart of the intestinal flora of grouper. A represents the blank control group, B represents the virus-infected group, and C represents the yolk antibody immersion treatment group. DETAILED DESCRIPTION In order to more clearly and thoroughly understand the technical solution of the present invention, the present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: Example 1 Expression of recombinant plasmid pET-28a-CP in E. coli: Add 1 μL of the recombinant plasmid to 25 μL of BL21 competent cells on ice and let it stand on ice for 30 min; after heat shock in a 42 °C water bath for 90 s, quickly transfer it to ice and let it stand for 10 min; add 40 μL of antibiotic-free LB liquid medium to the EP tube and incubate it in a 37 °C constant temperature shaker at 220 r / min for 20 min; spread all the bacterial solution evenly on the LB plate containing kanamycin antibiotic and incubate overnight for 16 h. Pick colonies of appropriate size and inoculate them into 1 L of LB liquid medium, add kanamycin antibiotic (1:1000), measure the OD value of the bacterial solution after shaking culture in a 37 °C shaker for 3 h, when the OD value is between 0.6 - 0.7, add IPTG with a final concentration of 0.3 mmol / L and culture it in a 15 °C shaker for overnight expression. Example 2 Purification of antigen protein: Resuspend the bacterial solution with Lysis Buffer (25 mM Tris-HCl (pH 7.5), 250 mM NaCl, 1 mM EGTA) and 1 mM PMSF, 4 mM β-ME solution, break the bacteria under high pressure of 750 bar for 15 min, centrifuge the suspension at 4 °C, 17000 rpm for 30 min, and collect the supernatant to extract the protein. Add the collected supernatant to the Ni column, adjust the flow rate of the liquid through the flow limiting valve to about 0.9 mL / min, after the supernatant completely flows through the Ni column, wash it in sequence with Lysis Buffer containing different concentrations of imidazole (0 mM, 20 mM, 50 mM, 300 mM), and the whole process is carried out in a low-temperature environment, such as Figure 1 As shown, the size of the finally eluted protein is 44 kDa. Concentrate and change the solution of the collected eluate containing 300 mM with a 30 kD concentrator tube to remove imidazole, and at the same time dilute it with Lysis Buffer without B-ME and PMSF (25 mM Tris-HCl, 250 mM NaCl; 1 mM EGTA), and repeat it three times. Put the diluted solution into a dialysis bag, place it in 500 mL of Lysis Buffer, add 500 mL of buffer1 (50 mM Tris-HCl, 250 mM NaCl, 750 mM (NH4)2SO4) drop by drop with a constant flow pump, discard half of the solution after completion, and then add 500 mL of buffer2 (20 mM Tris-HCl, 200 mM NaCl, 1% Glycerol, 2 mM CaCl2) drop by drop, and perform folding dialysis for 24 h. Collect the antigen protein after folding dialysis and transfer it to a 100 kD concentrator tube to concentrate it to 10 mg / mL, and perform size exclusion chromatography, using Superose TM6 The 10 / 300GL chromatography column was connected to the AKTA-pure system according to the operating requirements. First, it was equilibrated with GF Buffer (20 mM Tris, 200 mM NaCl, 2 mM CaCl2) for one column volume to make the solution environment in the column consistent with that of the protein sample. The antigen protein sample was injected into the sample loop with a syringe, and the running flow rate was set at 0.4 mL / min. Referring to Figure 2 chromatography, the protein eluting at 8 mL of column volume was collected, and the result was verified by Western blot as Figure 3 shown that the antigen protein obtained after folding dialysis treatment had higher purity than the protein without dialysis treatment. The antigen protein after dialysis was further verified, and its negative staining electron micrograph of the protein was as Figure 4 shown. The diameter of the pseudovirus particles assembled from the antigen protein was between 21 - 32 nm, presenting a uniform and clear spherical structure. Example 3 Immunization of laying hens: The antigen protein was fully emulsified with an equal volume of adjuvant. For the first immunization, the vaccine emulsified with the antigen protein and an equal volume of complete Freund's adjuvant was injected. For the second and third immunizations, the vaccine emulsified with incomplete Freund's adjuvant was used instead. The vaccine concentration was controlled at 0.3 mg / mL, and 1 mL of the vaccine was injected into each healthy hen, injected into both wings and the pectoral muscles on both sides, 0.25 mL was injected at each point, and immunization was carried out once every 2 weeks. Eggs were collected immediately after the first immunization as Figure 5 and stored at 4°C for later use. Example 4 Purification of egg yolk antibodies: The eggs were cleaned, disinfected with a benzalkonium bromide solution, the eggshells were wiped with 75% alcohol, then irradiated with an ultraviolet lamp for 30 min, air-dried, the eggshells were opened, the egg whites were separated, and the egg yolks were retained. The remaining egg white on the surface of the egg yolks was wiped clean with a sterilized filter paper. The egg yolk membrane was punctured with the tip of a syringe, and the egg yolk liquid was aspirated. Antibody extraction by ammonium sulfate stepwise precipitation: Dilute 1 part of egg yolk solution with 9 parts of deionized water according to a volume ratio of 1:9, and stir well. Adjust the pH of the egg yolk solution to 5.0 with dilute HCl, then mix evenly on a vortex shaker for 10 min, freeze overnight at -20 °C to defat, thaw the next day, and transfer the egg yolk dilution into a centrifuge tube. Centrifuge at 12000 r / min for 30 min at 4 °C, collect the supernatant, which is the water-soluble component of egg yolk antibody, add saturated ammonium sulfate to a final concentration of 50% (W / V) respectively, stir well to dissolve, then let stand at 4 °C for 2 h, centrifuge at 12000 r / min for 30 min at 4 °C, and discard the supernatant. Add an appropriate amount of pre-cooled PBS (pH 7.4) solution to the precipitate to suspend and dissolve it, then add saturated ammonium sulfate to a final concentration of 33% (W / V), mix evenly on a vortex shaker for 10 min, and let stand overnight at 4 °C. Centrifuge at 12000 r / min for 30 min at 4 °C, discard the supernatant, weigh the precipitate, dissolve the precipitate in PBS (pH 7.4) solution, transfer it to a dialysis bag, and dialyze overnight in ultrapure water. Finally, filter with a 0.22 μm filter membrane to obtain a purer egg yolk antibody, referring to Figure 6 The antibody extracted by ammonium sulfate precipitation is purer. Example 5 Determination of egg yolk antibody performance: (1) Detection of egg yolk antibody titer: Antigen coating: Dilute the antigen protein to 10 μg / mL with coating buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, dissolved in ultrapure water and made up to 1 L), add 100 μL per well to a 96-well plate, and incubate overnight at 4 °C in the refrigerator. Blocking: The next day, discard the coating solution in the well plate, wash 2 times with washing buffer (1 L of PBS, add 0.5 mL of Tween-20; 200 μL / well), then add 200 μL of blocking solution (1.5 g of bovine serum albumin, 2.5 g of skim milk powder, dissolved by stirring in PBS solution and made up to 50 mL) per well, incubate at 37 °C in an incubator for 1 h, discard the blocking solution, and wash 4 times (washing means adding the buffer, incubating at room temperature for 5 min, discarding the buffer, and patting dry on a paper towel before continuing the operation). Sample addition: Dilute the egg yolk antibody to be tested in a serial dilution with antibody diluent (0.05 g of BSA, 0.05 g of skim milk powder, dissolved in PBST solution and made up to 50 mL), with a gradient from 1:2000 to 1:65536000, incubate at 37 °C for 2 h, drain, and wash 4 times. Add enzyme-labeled secondary antibody: Dilute HRP-rabbit anti-chicken IgG with antibody diluent at a ratio of 1:5000, add 100 μL per well to each reaction well, incubate at 37 °C for 1 h, drain, and wash 4 times. The egg yolk antibody in the control group is used as a negative control. Color development: Add TMB color developer in the dark, 100 μL / well, gently tap to mix evenly, and develop color in the dark at 37 °C for 15 min. Termination: Add 50 μL of termination solution to each well, and detect OD450nm with an enzyme-linked immunosorbent assay (ELISA) reader. Result: When OD sample / OD negative ≥ 2.1, the maximum dilution multiple is the titer of the egg yolk antibody. The determination results of the antibody titer after the fourth immunization (the 9th week) are as Figure 7 , and the antibody titer value obtained is 1:16384000. The change rule of the egg yolk antibody with the immunization time of laying hens is as Figure 8 shown. Specific antibodies were detected in the eggs collected 1 week after the first immunization. The antibody level reached the peak at the 9th week and was still detectable at a relatively high level in the eggs until the 39th week after immunization, indicating that the antigen protein has a long immune response time in laying hens. (2) Specific detection of egg yolk antibody: Separate the capsid protein of grouper nervous necrosis virus by SDS-PAGE. After electrophoresis, cut the gel into an appropriate size, and soak it in the transfer buffer together with the nitrocellulose membrane and transfer filter paper for 15 min. Place it in the order of filter paper - gel - NC membrane - filter paper from bottom to top, with two filter papers on the top and bottom respectively. Remove air bubbles from each layer, set a constant voltage of 90 V, and transfer for 65 min. After completion, remove the membrane and wash it 3 times (wash for 10 min each time on a shaker), add the blocking solution (TBST containing 5% skim milk powder), and block at room temperature for 3 h. Incubation: Use the egg yolk antibody as the primary antibody, dilute it with the blocking solution at a ratio of 1:1000, incubate overnight, recover the primary antibody and store it at -20 °C, wash it 3 times with TBST, 10 min each time. Dilute the secondary antibody HRP-rabbit anti-mouse IgG with the blocking solution at a ratio of 1:5000, incubate at room temperature in the dark for 30 min, recover the secondary antibody and store it at -20 °C, wash it 4 times with TBST, 10 min each time. After washing with TBST, add the ECL color developer in the dark, and develop the image after incubating for 60 s. The results are as Figure 9 shown, verifying that specific egg yolk antibodies against grouper nervous necrosis virus can be obtained by the method described in the present invention. (3) Stability detection of egg yolk antibody: Investigate the effects of temperature, pH, pepsin, and seawater immersion on the egg yolk antibody. The results are as Figure 10 shown. The egg yolk antibody can maintain stable activity at temperatures below 70 °C, pH values in the range of 1 - 12, after pepsin treatment, and within 2 days of seawater immersion, fully meeting the application environment. Example 6 Preparation of egg yolk antibody lyophilized powder: AsFigure 11 As shown, the extracted yolk antibody presents a light yellow clear liquid. Placed in a 50 mL centrifuge tube, an appropriate amount of deionized water and a protective agent (chitosan) are added. After stirring with a magnetic stirrer, it is equilibrated at -4°C for 30 min, then slowly cooled to -20°C and frozen for 6 h, and finally placed at -80°C overnight. Then, a purified freeze-dried powder of yolk antibody against nervous necrosis virus is obtained by freeze-drying in a vacuum freeze-dryer at -80°C, with a chamber pressure of 1000 Pa for 48 h. At room temperature, the solid state of the antibody is a yellow mass or white powder (defatted). After the freeze-dried powder is reconstituted (it can dissolve in water), the antibody can be stably stored for a long time in both states. Example 7 Application of yolk antibody - Viral challenge protection test for larvae of Epinephelus coioides: Sixty larvae of Epinephelus coioides with a body weight of about 0.8 g were selected as Figure 12 , 15 in a group. In the water bodies of the first and second groups, yolk antibody against nervous necrosis virus (45 and 60 mg / L respectively) was added and continuously soaked for one week. The third and fourth groups were not treated. After one week, the first to third groups of Epinephelus coioides were challenged with the virus and the temperature was raised. One Epinephelus coioides injected intraperitoneally with an equal amount of nervous necrosis virus (0.2 mL / tail) was added to each group. The fourth group was not challenged. Other conditions remained unchanged. The status of Epinephelus coioides was observed every day, and the survival of fish in each group within one week was recorded; the brain and eye tissue sections of Epinephelus coioides in the fourth blank control group, the third virus-infected group, and the first antibody-soaked group were observed to investigate the protective effect of the yolk antibody on the brain and eyes; the intestinal flora of Epinephelus coioides in different groups was also measured to investigate the effect of the yolk antibody on the intestinal flora. As Figure 13 shown, by counting the survival results of fish in each group, the survival rate of Epinephelus coioides soaked with yolk antibody was 100%, and the survival rate of Epinephelus coioides in the control group not soaked with yolk antibody was 66.67%. As Figure 14 shown, through systematic histopathological analysis, it was observed that compared with Epinephelus coioides soaked with yolk antibody, a large number of vacuoles appeared in the eye and brain parts of Epinephelus coioides infected with nervous necrosis virus. The number of vacuoles in the fish soaked with yolk antibody was greatly reduced, and there was basically no difference from the normal group. It shows that the antibody has a certain protective effect on the fish and can reduce the damage of nervous necrosis virus to the larvae of Epinephelus coioides. As Figure 15As shown, the intestinal flora in the virus-infected group exhibited significant pathogenicity and environmental adaptability characteristics: the proportion of potential pathogenic bacteria increased significantly compared with the blank control group and the yolk antibody immersion group, and the proportion of pressure-resistant bacteria was significantly higher than that of other groups, indicating that the oxidative stress caused by virus infection selectively promoted the proliferation of pressure-resistant bacteria. The yolk antibody immersion group maintained the microbial community homeostasis through a dual mechanism: on the one hand, it inhibited aerobic pathogenic bacteria, and on the other hand, it promoted the increase in the proportion of anaerobic probiotics, which may be related to the neutralization of virus capsid proteins by yolk antibodies and the improvement of the intestinal microenvironment. As described above, the above are only the preferred embodiments of the present invention, and do not impose any formal limitations on the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the thinking essence and technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A grouper nervous necrosis virus capsid protein antigen, characterized in that: The gene sequence of the antigen is shown as SEQ ID NO.

1.

2. Use of the antigen according to claim 1 in the preparation of a drug for preventing and treating nervous necrosis virus.

3. An egg yolk antibody against grouper nervous necrosis virus, characterized in that: The yolk antibody is obtained by purifying the yolk of the eggs laid by laying hens immunized with the antigen expressed after recombination of the gene sequence shown in SEQ ID NO.1 of claim 1.

4. Use of the yolk antibody according to claim 3 in the preparation of a drug for preventing and treating nervous necrosis virus.

5. The preparation method of the yolk antibody against grouper nervous necrosis virus according to claim 3, wherein, The method steps are as follows: S1. Antigen protein expression: Recombine the grouper nervous necrosis virus capsid protein coding gene fragment shown in SEQ ID NO.1 into the pET-28a vector to construct the pET-28a-CP recombinant plasmid, and introduce it into Escherichia coli competent cells for expression; S2. Antigen protein purification: Expand the culture of Escherichia coli competent cells, and after disruption, affinity chromatography, concentration, folding dialysis, and size exclusion chromatography, obtain a high-concentration antigen protein; S3. Laying hen immunization: Emulsify the antigen protein prepared in step S2 with an adjuvant into a vaccine, inject it into healthy hens, and collect their eggs; S4. Yolk antibody purification: Clean and disinfect the eggs, separate the yolks, suck out the yolk liquid, and obtain the yolk antibody through purification.

6. The preparation method of the yolk antibody of grouper nervous necrosis virus according to claim 5, characterized in that: The process of folding dialysis in step S2 is to gradually add buffer1 drop by drop at a flow rate of 1.5 - 2.5 mL / min with a constant flow pump in a low-temperature environment. After the addition is completed, discard half of the solution, and then add buffer2 drop by drop at a flow rate of 0.5 - 1.5 mL / min.

7. The preparation method of the yolk antibody of grouper nervous necrosis virus according to claim 6, characterized in that: The buffer1 is a mixed solution of Tris-HCl, NaCl, and (NH4)2SO4; the buffer2 is a mixed solution of Tris-HCl, NaCl, Glycerol, and CaCl2.

8. The preparation method of the yolk antibody of grouper nervous necrosis virus according to claim 5, characterized in that, The column for size exclusion chromatography in step S2 is Superose TM 6 Increase 10 / 300GL column.

9. The preparation method of the yolk antibody of grouper nervous necrosis virus according to claim 5, characterized in that, The purification method in step S4 is the ammonium sulfate precipitation method.

10. Use of the yolk antibody of grouper nervous necrosis virus prepared by the preparation method according to any one of claims 5 - 9 in the preparation of a drug for preventing and treating grouper viral nervous necrosis.

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