Neutralizing epitope polypeptide of grouper nervous necrosis virus and application of neutralizing epitope polypeptide in serological diagnosis test paper

Abstract

The invention provides a neutralizing epitope polypeptide of grouper nervous necrosis virus and application of the neutralizing epitope polypeptide in serological diagnosis test paper, and the amino acid sequence of the neutralizing epitope polypeptide is SEQ ID NO: 1 or SEQ ID NO: 2. On the other hand, the invention also provides a serological diagnosis test strip for the nervous necrosis virus disease of the grouper, wherein the NNV capsid protein neutralizing epitope polypeptide is used as a detection line. The antigen polypeptide used by the test strip is an NNV capsid protein neutralizing epitope specific fragment, the antigen recognition effect is improved by optimizing the mixing ratio, cross reaction with other pathogen positive serum of grouper is avoided, and the detection accuracy is ensured.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of detection and diagnosis of aquatic animal diseases, and particularly relates to identification of a grouper nervous necrosis virus neutralizing epitope polypeptide and application thereof in a serological diagnosis test paper for the disease. BACKGROUND

[0002] Viral nervous necrosis is an important epidemic disease that seriously endangers the fish farming industry, and the infection rate and mortality rate of its pathogen, nervous necrosis virus (NNV), can be as high as 100%. In particular, the infection of juvenile and young fish is serious. Since the late 20th century, NNV has been reported in cultured or wild Epinephelus coioides, Epinephelus fuscoguttatus, Epinephelus lanceolatus♂ × E. fuscoguttatus♀ Paralichthys olivaceus Cynoglossus semilaevis Gunther Gadus macrocephalus , etc. in China, causing huge economic losses to the aquaculture industry in China.

[0003] The symptoms of fish infected with NNV are a large number of vacuolization necrosis in the nervous system and retina, so it is also known as viral encephalopathy and retinopathy (VER). Fish infected with the virus move slowly; the swimming posture is abnormal, such as spiral swimming, circular swimming, and rotating swimming; the eyeball protrudes, etc.; upon dissection of the diseased fish, the liver presents a soil yellow color and atrophy symptoms; the kidney organ is eroded and necrotic, the brain is hemorrhagic, and the swim bladder is swollen and pale in color. The clinical symptoms progress rapidly and are extremely easy to cause large-scale outbreaks.

[0004] Capsid protein (CP) is the only structural protein of NNV, and multiple antigen epitopes are distributed on the CP, among which the antigen epitopes that can be specifically recognized by antibodies with virus neutralizing activity are called neutralizing epitopes. Neutralizing epitopes are usually located in the surface exposed region of CP, have good immunogenicity, and can induce the host to produce high-affinity neutralizing antibodies. Neutralizing epitopes not only have important value in vaccine design, but also provide a key target for the detection of specific antibodies and antigens. Through bioinformatics analysis and experimental verification, neutralizing epitopes and their amino acid sequences can be identified, and neutralizing epitope polypeptides can be artificially synthesized as antigen components in diagnostic reagents. The polypeptides have the advantages of clear structure, good stability, and strong immunospecificity, and can significantly improve the accuracy and reliability of detection.

[0005] ​​​The application of serological techniques to detect NNV-specific antibodies can effectively diagnose the infection state of fish, and the level of specific antibodies can also determine the immune effect of fish after vaccination, providing an important basis for the effective prevention and control of diseases. Among them, colloidal gold immunochromatography technology is a rapid diagnostic method based on the specific binding reaction of antigen and antibody, which has been widely used in the preliminary screening of animal diseases and human diseases. This technology has good sensitivity and specificity, does not require a laboratory environment, professional equipment and personnel operation, and has a simple operation process, which can meet the on-site detection needs of rapid and convenient diagnosis of primary breeding farms.

[0006] Therefore, by identifying and screening the neutralizing epitope in the NNV capsid protein, using it as the detection antigen of the serological diagnostic test strip, developing a serological diagnostic test strip suitable for on-site rapid detection of grouper NNV-specific antibodies, not only can the infection or immune state of fish be determined immediately, but also provides scientific and convenient technical support for vaccine immune effect evaluation, breeding management decision-making and disease early warning. SUMMARY

[0007] The present application aims to provide a grouper NNV neutralizing epitope polypeptide, and a preparation method and application of a nervous necrosis virus disease serological diagnostic test strip based on the polypeptide. Based on obtaining an efficient neutralizing antigen epitope polypeptide, the present application constructs a test strip for detecting whether the serum of a grouper contains NNV-specific antibodies. The test strip constructed by the present application has the advantages of simple operation, no need for instruments, and being suitable for on-site rapid detection, and can complete the detection within 10 min, meeting the needs of on-site diagnosis of the disease.

[0008] The present application first provides a NNV capsid protein neutralizing epitope polypeptide, which is polypeptide CP-I, and the amino acid sequence thereof is QYTRTLLWTSSG (SEQ ID NO: 1); The present application further provides another NNV capsid protein neutralizing epitope polypeptide, which is polypeptide CP-II, and the amino acid sequence thereof is NTDVVNVSVLCR (SEQ ID NO: 2); The present application further provides a use of the NNV capsid protein neutralizing epitope polypeptide, which is in the preparation of an antibody.

[0009] As a specific description of an embodiment, the antibody is a polyclonal antibody.

[0010] The present application further provides another use of the NNV capsid protein neutralizing epitope polypeptide, which is used as a detection antigen for preparing a product for detecting NNV-specific antibodies of a grouper; As a specific description of an embodiment, the product is a grouper nervous necrosis virus disease serological diagnostic test strip, which can be used to determine whether a grouper is infected with NNV.

[0011] The application further provides a serological diagnostic test strip for nervous necrosis virus disease of grouper, wherein the detection line uses the neutralizing epitope polypeptide of the NNV capsid protein. Furthermore, the neutralizing antigen epitope polypeptide is a mixed polypeptide of CP-I and CP-II, and the content ratio is preferably 2:1.

[0012] Furthermore, the gold-labeled antibody on the serological test strip is a rabbit anti-grouper IgM antibody.

[0013] The quality control line (C line) of the test strip is coated with a goat anti-rabbit IgG secondary antibody, which is used to verify the effectiveness of the test strip.

[0014] Compared with the prior art, the application has the following beneficial effects: (1) The antigen polypeptide used in the test strip is a neutralizing epitope specific fragment of the NNV capsid protein, and the recognition effect of the antigen is improved by optimizing the mixing ratio, and there is no cross reaction with other antigen positive serum of grouper, thereby ensuring the accuracy of the detection.

[0015] (2) No complex experimental steps and professional technicians are required, and it is suitable for primary personnel to use; no instrument is required, and the detection process does not depend on any precise instrument equipment, which is convenient for carrying out in the breeding field; the operation is simple, only the serum sample needs to be diluted and added to the sample pad, and then the result can be directly judged, thereby reducing the operation threshold; (3) The application has the advantage of visualization, and the detection result can be judged by directly observing the color development result of the test strip with the naked eye. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 : SOPMA prediction of the secondary structure of the NNV capsid protein; Figure 2 : DNA star software prediction of the secondary structure of the NNV capsid protein; Figure 3 : IEDB software prediction of the B cell linear antigen epitope of the NNV capsid protein; Figure 4 : Dot-blot identification of the specificity of the neutralizing epitope polypeptide of the NNV capsid protein, wherein Fish 1-Fish 6 are the sera of different grouper individuals infected with NNV; Figure 5 : Figure A is a microscopic observation of the effect of different treatments on the development of CPE induced by RGNNV infection, Figure B is a CCK-8 analysis of the effect of different pre-incubation groups on the cell viability of RGNNV infection, Figure C is the effect of different pre-incubation groups on the virus titer, and Figure D is a qPCR analysis of the effect of different pre-incubation groups on the viral replication level. cpEffect of gene replication, Figure E is a Western blot analysis of the effect of different pre-incubation groups on the expression level of CP, CP-I and CP-II are CP-I polyclonal antibody and CP-II polyclonal antibody pre-incubation groups, PC is a virus without antibody pre-incubation group, and NC is a culture medium without virus and antibody pre-incubation group; Figure 6 : SDS analysis of IgM purity extracted from grouper serum, the figure shows that lane M is Marker, lane 1 is unpurified grouper serum, and lane 2 is purified IgM; Figure 7 : Western-blot result of rabbit anti-grouper IgM polyclonal antibody and grouper serum, wherein M is a protein relative molecular mass standard, and lane 1 is purified grouper serum IgM; Figure 8 : Determination of the optimal labeling conditions of gold-labeled antibodies by OD value method, wherein Figure A is the determination of the optimal labeling pH value, and Figure B is the determination of the optimal protein amount for labeling; Figure 9 : Determination of the optimal mixing ratio of test line antigens; wherein A is healthy grouper serum; B-D are CP-I and CP-II with mixing ratios of 1:2, 1:1 and 2:1, respectively; Figure 10 : Schematic diagram of test strip assembly and reaction principle; Figure 11 : Test strip result determination diagram; Figure 12 : Specific detection result diagram of test strip, wherein A is healthy grouper serum, B is NNV positive serum of grouper, C is Vibrio harveyi positive serum of grouper, D is Vibrio parahaemolyticus positive serum of grouper, and E is Vibrio alginolyticus positive serum of grouper; Figure 13 : Sensitivity test result diagram of test strip, test strips (B-H) are added with NNV positive serum of grouper with titers of 1:10, 1:50, 1:100, 1:200, 1:400, 1:800 and 1:1600, respectively, and A is healthy grouper serum. DETAILED DESCRIPTION

[0017] The detection principle of the serum diagnostic test strip for grouper nervous necrosis virus disease used in the application is as follows: The NNV antibody in the sample binds with the colloidal gold labeled rabbit anti- grouper IgM antibody to form a "colloidal gold labeled rabbit anti-grouper IgM antibody- NNV antibody" complex; when the complex is chromatographed to the T line, the NNV antibody binds with the coated polypeptide to form a "polypeptide-NNV antibody-colloidal gold labeled rabbit anti-grouper IgM antibody" sandwich structure, and the T line develops color (red); the free colloidal gold labeled rabbit anti-grouper IgM antibody continues to migrate to the C line, binds with the goat anti-rabbit IgG to develop color, proving that the test strip has effectiveness.

[0018] Another aspect of the present application also provides a preparation method of the grouper nervous necrosis virus disease serological diagnostic test strip. The colloidal gold particles with a diameter of about 20 nm are prepared by a sodium citrate reduction method, and the labeling conditions of the colloidal gold and the rabbit anti-grouper IgM polyclonal antibody are determined by an OD value method. The optimal labeling pH value is 9.2, and the optimal labeling concentration of the rabbit anti-grouper IgM polyclonal antibody is 7.5 μg / mL. The optimal dilution degree of the gold-labeled antibody is 1:10, the concentration of the two NNV capsid protein neutralizing epitope polypeptides is 2 mg / ml, and the optimal mixing ratio of CP-I and CP-II is 2:1. The colloidal gold labeled rabbit anti-grouper IgM antibody is sprayed on a glass fiber membrane to prepare a gold-labeled pad, and the polypeptide and the goat anti-rabbit IgG are streaked on a nitrocellulose membrane as a detection line and a quality control line, respectively. The sample pad, the absorption pad and the PVC bottom plate are assembled into the grouper nervous necrosis virus disease serological diagnostic test strip. The test strip has a positive reaction with the serum of the grouper infected with NNV, the highest dilution degree of the detected serum is 1:200, and there is no cross reaction with other pathogenic antisera, indicating that the test strip has good specificity and sensitivity. The test strip can be sealed and stored at room temperature for 5 months and at 4°C for 10 months, and has good stability.

[0019] The present application will be further described below by combining the accompanying drawings and through specific examples.

[0020] Example 1: Screening of NNV capsid protein neutralizing epitope polypeptide The structure of CNPgg2018 capsid protein (GenBank Accession No. QOV05415.1) was analyzed by using SOPMA (https: / / npsa-prabi.ibcp.fr / ) online prediction software and DNAStar software. The parameters mainly included alpha helix (Alpha, Amphipathic Regions-Eisenberg), beta-turn (Beta, Amphipathic Regions Eisenberg), random coil, flexibility (Flexible Regions - Karplus-Schulz), antigenicity (Antigenic Index - Jameson-Wolf) and surface accessibility (Surface Probability Plot - Emini). According to the analysis of the secondary structure of NNV capsid protein by SOPMA online software, the results are shown in Figure 1 As shown, the random coil (Cc) in the secondary structure of NNV capsid protein accounted for 57.99%, the extended chain (Ee) accounted for 27.51%, the alpha-helix (Hh) accounted for 10.36%, and the beta-turn (Tt) accounted for 4.14%; according to the analysis of the secondary structure of NNV capsid protein by Protean program in DNA star software, the results are shown in Figure 2 The potential epitopes located in the beta-turn and random coil regions were screened by considering the surface accessibility index, antigenicity index, flexibility and hydrophobicity. The NNV capsid protein antigenic epitopes were further predicted by using IEDB (http: / / tools.immuneepitope.org / main / ) online prediction software, and the results are shown in Figure 3 As shown, the regions with higher comprehensive scores of B cell linear antigenic epitopes were selected as candidate polypeptide sequences. By comprehensively analyzing the prediction results of the two software, nine possible NNV capsid protein polypeptide sequences were selected (Table 1).

[0021] Table 1: NNV capsid protein B cell antigenic epitope table

[0022] Finally, according to the difficulty of artificial synthesis of polypeptide and the degree of conjugation with KLH, the NNV capsid protein polypeptide sequences were selected as CP-I: QYTRTLLWTSSG; CP-II: NTDVVNVSVLCR.

[0023] Dot-blot method was used to identify the synthetic polypeptides. First, the polypeptides were spotted on the surface of nitrocellulose membrane and naturally dried at room temperature. Then the membrane was blocked in 5% BSA to block non-specific binding sites. After blocking, the membrane was incubated with the appropriate dilution of NNV-infected grouper serum as the primary antibody, and the unbound primary antibody was removed by washing. Then the membrane was incubated with the appropriate dilution of mouse anti-grouper IgM as the secondary antibody, and the unbound secondary antibody was removed by washing. Then the enzyme-labeled tertiary antibody was added for further incubation. After washing, the color developing substrate was added for color development reaction, and the results were finally interpreted by visual observation or imaging system. Positive results formed clear spots at the sample site. Figure 4 ).

[0024] In vitro neutralization experiment: After healthy Balb / c mice were raised in the laboratory for one week, two of them were selected and immunized with 1 mg / ml CP-I and CP-II polypeptides as antigens. The first time, the polypeptide suspension was mixed with an equal volume of Freund's complete adjuvant, and each mouse was injected intraperitoneally with 200 μl. Two weeks later, the same volume of polypeptide suspension was mixed with Freund's incomplete adjuvant, and each mouse was injected intraperitoneally with 200 μl for the first booster. Then, 200 μl of polypeptide suspension was injected into the tail vein every week. Seven days after the last immunization, the mice were anesthetized with ether, and the eyeball was removed to collect blood. The blood was placed at room temperature for 2 h and then placed in a 4 ℃ refrigerator overnight. The next day, the blood was centrifuged in a 4 ℃ centrifuge (10000g, 5 min). Finally, the obtained antiserum was aliquoted, and two polyclonal antibodies were obtained and stored at -80 ℃ for future use.

[0025] The RGNNV suspension with MOI=1 was aliquoted into two 1.5 mL centrifuge tubes, and CP-I and CP-II polyclonal antibodies were added, respectively. The positive control group (containing only RGNNV suspension without antibody) and the negative control group (containing only medium without virus and antibody) were set on a rotary incubator at 4 ℃ for 1 h. SSN-1 cells were inoculated in 6-well plates or 96-well plates, and when the cell confluence reached more than 80%, the virus-antibody complex was added and incubated at 28 ℃ for 1 h. After washing the cells with PBS for 3 times to remove the unbound virus, fresh L-15 medium containing 2% FBS was added for further culture for 3 days. During this period, the development of cytopathic effect (CPE) was observed and recorded under a microscope every day. On the last day of infection, the virus suspension containing cell debris in the 6-well plate was collected and freeze-thawed three times to calculate the virus titer and cell survival rate.

[0026] To further validate the neutralizing ability of CP-I and CP-II polyclonal antibodies, the RGNNV suspension at an MOI of 1 was incubated with CP-I and CP-II polyclonal antibodies and then inoculated onto SSN-1 cells in 6-well plates. Three days after infection, the cells were washed once with PBS, scraped with a cell scraper, and divided equally. Total RNA from one cell was extracted with TRIzol, converted to cDNA, and analyzed by qPCR. cp Another cell was lysed with RIPA buffer to extract total protein, and the relative expression of CP was analyzed by Western blot.

[0027] SSN-1 cells were infected with RGNNV pre-incubated with different antibodies. The results showed that after 3 days of infection, the positive control showed typical cytopathic effect, which was manifested by cell rounding, brightening, shrinkage, and aggregation into grape-like clusters. In contrast, only a small amount of corresponding cytopathic effect and cell death occurred in the CP-Ⅰ and CP-Ⅱ polyclonal antibody pre-incubation groups; and no CPE was observed in the negative control group ( Figure 5 -A); At the same time, the cell viability and RGNNV titer of each group were measured 3 days after infection. The results showed that after pre-incubation with CP-Ⅰ polyclonal antibody and CP-Ⅱ polyclonal antibody, the cell survival rate of SSN-1 cells was 100% ( Figure 5 -B). Similarly, pre-incubation with CP-Ⅰ polyclonal antibody and CP-Ⅱ polyclonal antibody reduced the amount of RGNNV in the culture supernatant ( Figure 5 -C); qPCR results showed ( Figure 5 -D), CP-Ⅰ polyclonal antibody and CP-Ⅱ polyclonal antibody pre-incubation can reduce the viral cp Copy number; Western blot results showed ( Figure 5 -E), the relative expression of CP decreased in the CP-Ⅰ and CP-Ⅱ polyclonal antibody pre-incubation groups. In summary, the polyclonal antibodies induced by CP-Ⅰ and CP-Ⅱ polypeptides can effectively neutralize RGNNV in vitro and block its infection and replication process.

[0028] Example 2 Preparation of rabbit anti-grouper IgM polyclonal antibodies 1. Purification of grouper serum IgM After the grouper serum was purified by Protein L affinity chromatography and molecular sieve, the unpurified grouper serum and the extracted IgM were simultaneously detected by SDS-PAGE ( Figure 5 ), the results showed that the extracted IgM had obvious bands at approximately 75 kDa and 27 kDa. The molecular weight indicated by these bands was consistent with the molecular weight of the IgM heavy chain and light chain. The bands were clear and there were few miscellaneous bands. This showed that the IgM obtained by the above purification effectively removed impurities, indicating that the purified IgM had a high purity and could provide reliable experimental materials for subsequent animal immunization experiments.

[0029] 2. Preparation of rabbit anti-Epinephelus malabaricus IgM polyclonal antibody Preparation of rabbit anti-Epinephelus malabaricus IgM polyclonal antibody: After the purchased New Zealand white rabbits were temporarily raised in the laboratory for one week, the purified Epinephelus malabaricus IgM was adjusted to a concentration of 1 mg / ml, and the New Zealand white rabbits were immunized subcutaneously at six points. In the first week, the purified IgM was mixed with an equal volume of complete Freund's adjuvant, and each inoculation was 600 μl. In the following two weeks, the purified IgM protein was mixed with an equal volume of incomplete Freund's adjuvant. In the last week, 600 μl of the purified Epinephelus malabaricus suspension was carefully injected into the rabbit's ear margin by ear margin intravenous injection. After one week, the New Zealand white rabbits were anesthetized with ether, and 10 ml of blood was drawn from the heart. The drawn blood was placed in a pre-autoclaved 10 ml centrifuge tube, and the tube was placed on a flat surface at room temperature for 2 h and then cooled overnight in a 4°C refrigerator. The next day, the centrifuge tube was placed in a 4°C centrifuge, centrifuged at 8000g for 30 min, and the supernatant was carefully aspirated and centrifuged for another 20 min. Finally, the obtained antibody was aliquoted and stored in a -80°C refrigerator for future use.

[0030] An equal amount of purified Epinephelus malabaricus IgM and 2xLoading buffer were mixed in a 1.5 ml centrifuge, heated in boiling water for 5 min, and then added to the holes of the SDS-PAGE electrophoresis plate together with the rainbow marker and the cooked sample. Each electrophoresis plate hole was added with 10 μl of sample. After electrophoresis, the length and width of the separation gel were measured with a ruler, and PVDF membranes of the same size were cut. The PVDF membranes were soaked in methanol for 30-60 s, then in ultrapure water for 2 min, and then in the transfer buffer. The filter paper, sponge pad, PVDF membrane, and SDS-PAGE gel were thoroughly soaked in the electrotransfer buffer. The black clamp, sponge pad, filter paper, PVDF membrane, gel, filter paper, sponge pad, and red clamp were arranged in the order of bottom to top, one layer after another, to form a "sandwich" structure, avoiding the generation of bubbles. After fixing the clamp, the black clamp was aligned with the positive electrode, and the red clamp was aligned with the negative electrode. The protein was transferred at a voltage of 30 V for 90 min.

[0031] After the transfer was completed, the gel was discarded, and the rainbow protein marker and sample lanes on the PVDF membrane were cut off with a knife. The PVDF membrane was placed in a 1x protein-free fast blocking solution for blocking for 15 min. The PVDF membrane was washed with a PBST eluent containing 0.5% Tween-20 for three times in a shaker for 5 min each time. The prepared polyclonal antibody was diluted 10,000 times to incubate the PVDF membrane, and rabbit negative serum was used as a negative control band and placed in a shaker for incubation for 1 h. The PBST solution was eluted for three times for 5 min each time. HRP-conjugated goat anti-rabbit IgG was diluted 30,000 times to incubate the PVDF membrane for 45 min, and the PBST solution was eluted for three times. The color developing solution was prepared in advance, the PVDF membrane was placed in a chemiluminescence instrument, the luminescent solution was added to the PVDF membrane, the exposure time of the chemiluminescence instrument was adjusted, and a photograph was taken for record.

[0032] The results of the Western blot analysis Figure 6 ) showed that the rabbit anti-Epinephelus coioides IgM polyclonal antibody could specifically recognize IgM, and clear bands were emitted at positions of about 75 kDa and 27 kDa, which were consistent with the molecular weights of IgM heavy chains and light chains, proving that the obtained polyclonal antibody had good specificity and could be used for subsequent gold-labeled antibody preparation research.

[0033] Example 3: Preparation of a test strip for serological diagnosis of Epinephelus coioides nervous necrosis virus disease 1. Solution preparation Gold label buffer: 0.1 M boric acid (pH=8.5) buffer Gold label pad treatment solution: 100 ml of 0.1 M boric acid (pH=8.5) buffer was weighed, 1 g of BSA, 2.5 g of sucrose, and 0.2 ml of Tween-20 were added to a container, and after stirring uniformly, it was stored at 4°C for standby use.

[0034] 2. Preparation of colloidal gold The glass used in the preparation of colloidal gold is soaked in acid alcohol for two days. A 50 ml beaker, a 50 ml graduated cylinder, and acid alcohol are used. After soaking, the glass is cleaned with tap water, pure water, and ultrapure water three times, and then sealed with plastic wrap and placed in an oven to dry. The rotor is washed with ultrapure water and placed in a beaker. Then 49.5 ml of ultrapure water is added to the beaker, followed by 500 μl of 1% HAuCl4, and the liquid level is marked with a marker. To replenish the water to the original volume after preparation, place it on a magnetic stirrer and stir at low speed. The stirring speed should not be too high during the gold preparation process, as it will affect the quality of the prepared colloidal gold. Turn on the heating mode of the magnetic stirrer and stir while heating until boiling. This process takes about 10 minutes. Once boiling begins, quickly add 750 μl of 1% sodium citrate (Na3C6H5O7·2H2O) and continue heating and stirring. The color will change from light yellow to gray, then to black, and finally to wine red. The color change process will last for about 1 minute. When the color stabilizes at wine red, turn off the heating mode and continue stirring for 2-3 minutes. Stop stirring and cool at room temperature.

[0035] 3. Preparation of gold-labeled antibody Determination of the optimal gold labeling pH: Take 5 centrifuge tubes and add 2 mL of colloidal gold solution to each tube. Set 5 different pH gradients (6, 7, 8, 9, and 10). Adjust the pH with 0.1 mol·ml -1 of potassium carbonate solution and 0.1 mol·ml -1 of hydrochloric acid. Then add 100 μl of antibody to each test tube and mix quickly. Shake for 10 minutes and then let stand for 30 minutes. Add 10 μl of 10% NaCl solution and mix well. Let stand for 2 hours. Measure the OD value at 520 nm under different pH values using a spectrophotometer. Plot the OD value curve with the OD value as the vertical coordinate and the pH value as the horizontal coordinate. Record the pH value corresponding to the maximum OD value as the optimal labeling pH value. Repeat the experiment with pH values of X-0.7, X-0.2, X, X+0.2, and X+0.7.

[0036] Determination of the optimal antibody amount for gold labeling: Take 9 centrifuge tubes and add 2 mL of colloidal gold solution to each tube. Adjust the pH with 0.1 mol·ml -1 of potassium carbonate solution and 0.1 mol·ml -1The pH value is adjusted by hydrochloric acid to adjust the pH value of colloidal gold to the optimum; 9 different concentration gradients of polyclonal antibody are set (2.5 μg, 5 μg, 7.5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg), mixed, and left to stand at room temperature for 30 min, then 10 μl of 10% NaCl solution is added, mixed, and left to stand for 2 h; the OD value at 520 nm under different concentrations of antibody labeling is determined by spectrophotometry, and the OD value curve is drawn, with the antibody concentration as the abscissa and the OD value as the ordinate, and the antibody concentration corresponding to the maximum OD value is recorded as the optimal labeled protein amount.

[0037] The OD value at 520 nm of the gold-labeled antibody obtained at different pH values is different, and the pH value corresponding to the highest OD value is the optimal gold labeling pH value. The pH value-OD520 value curve shows that the pH value corresponding to the highest OD value is 9.2, and thus the optimal labeling pH value is determined to be 9.2. Figure 7 The concentration of rabbit anti-Epinephelus malabaricus IgM polyclonal antibody-OD520 value curve shows that the concentration corresponding to the highest OD value is 7.5 μg / mL, and thus the optimal labeling concentration is determined to be 7.5 μg / mL. Figure 7

[0038] The rabbit anti-Epinephelus malabaricus IgM polyclonal antibody with the highest titer, the best effect, and high purity is selected as the gold-labeled antibody for labeling, and 50 ml of colloidal gold solution is measured in a beaker, 0.1 mol·ml -1 The pH value is adjusted to 9.2 by potassium carbonate solution, and the solution is placed on a magnetic stirrer for stirring, then a proper amount of purified polyclonal antibody is added dropwise, stirred for 20 min, and left to stand for 10 min. The volume of 5% BSA to be added is calculated to make the final concentration of BSA 1%, stirred for 15 min, and left to stand for 10 min. The role of BSA is to block non-specific sites and increase stability. The gold-labeled antibody solution is poured into a centrifuge tube, centrifuged at 1200 rpm for 20 min, and the aggregates are removed, and the supernatant is collected. The centrifuge is pre-cooled, the gold-labeled antibody solution is centrifuged at 6000 rpm at 4°C for 30 min, the supernatant is discarded, and the precipitate is collected, and the operation should be gentle, and the precipitate is resuspended with 0.1M boric acid (pH=8.5) washing solution to make the final volume 10% of the original volume. The precipitate is resuspended with 0.1M boric acid (pH=8.5) under the above conditions, and stored at 4°C for standby.

[0039] 4. Selection of gold-labeled pad treatment solution ​Selection of gold label pad treatment liquid, four kinds of gold label pad treatment liquid formula were determined, solution preparation method as shown in Table 2. The appropriate size of glass fiber was immersed in the treatment liquid, 2 h, freeze-dried for 2 h, gold label antibody was diluted 1:10, sprayed on the glass fiber to prepare gold label pad. Then assemble the test strip, observe the effect of gold label pad releasing gold label antibody through experiment.

[0040] Table 2: Screening table of gold label pad treatment liquid

[0041] The gold label pad was prepared with different gold label pad treatment liquid, and the test strip was assembled with it, and the serum of Epinephelus was added dropwise. It was found that the gold label pad treatment liquid I could effectively promote the release of gold label antibody, and the detection result did not appear false positive, so the glass fiber membrane was treated with it to prepare the gold label pad (Table 3).

[0042] Table 3: Result table of different gold label pad treatment liquid

[0043] 5. Preparation of NC membrane The concentrations of CP-I and CP-II were both 2 mg / ml, CP-I and CP-II were mixed in the proportion of 1:2, 1:1 and 2:1, respectively, and were drawn on the NC membrane as the detection line. The concentration of goat anti-rabbit IgG was adjusted to 0.5 mg / ml, and was drawn on the quality control line of the NC membrane with a membrane drawing instrument. The nitrocellulose membrane, water absorption pad, gold label pad and nitrocellulose membrane were pasted on the PVC bottom plate in turn, and the test strip was cut into 3.7 mm with a cutting machine and put into the card shell. The serum of Epinephelus was added dropwise at the sample site, and the best drawing ratio of the drawn antibody was determined by color development effect.

[0044] The results showed (as shown in Figure 8 ), after adding the healthy serum of Epinephelus, only the quality control line developed color, and the test strip result showed negative; after adding the positive serum of Epinephelus, when the mixing ratio of CP-I and CP-II was 1:2, the color band showed lighter; when the ratio was 1:1, the color of the color band was deeper; when the ratio of the antibody was 2:1, the color development effect was the best, so the ratio of 2:1 was used as the detection line drawn on the NC membrane.

[0045] 6. Assembly of test strip Assembly of test strip: During the assembly of test strip, gloves were required throughout the process, and the assembly process was as follows: Figure 9The assembly of the test strip is shown, and special attention should be paid to the connection between the components. The connection needs to be tight, and the overlap is about 2 mm, which is beneficial to maintain the stability of the test strip detection. After assembly, cut it into a test strip with a width of 3.7 mm in a cutting machine, and then put it into a card shell. Then, with a desiccant, a test tube containing 0.1 M boric acid buffer (pH=8.5), and a sample suction tube, put them into an aluminum foil bag and seal and store at 4°C.

[0046] Result interpretation Figure 10 ): Dilute the grouper serum 10 times, and add 100 μl to the sample hole. After 10 minutes of reaction, observe the results. The detection line and the control line are colored, and the detection result is positive. The detection line is not colored, and the control line is colored, and the detection result is negative. If the control line is not colored, regardless of whether the detection line is colored or not, the result is invalid, indicating that the test strip has failed and needs to be retested or the quality of the test strip needs to be checked.

[0047] Example 4: Specificity verification of test strip During the test strip detection process, different types of grouper antisera were used, including grouper NNV positive serum, lymph cyst virus positive serum, Vibrio alginolyticus positive serum, Vibrio harveyi positive serum, and Vibrio parahaemolyticus positive serum, to evaluate whether the test strip has cross-reaction with other pathogen sera. Add the serum to the sample hole of the test strip, and after 10 minutes of reaction, observe and record the color development. The results show that Figure 11 ), only when the grouper NNV positive serum is added, the detection line and the control line of the test strip are colored positive; while adding other positive sera, only the control line of the test strip is colored, and the detection line is not colored negative; when adding healthy grouper serum, only the control line is colored, and the result is negative, indicating that the test strip has no cross-reaction with other positive sera, and has good specificity.

[0048] Example 5: Sensitivity verification of test strip To analyze the sensitivity of the test strip, the grouper NNV positive serum was diluted by 1:10, 1:50, 1:100, 1:200, 1:400, 1:600, and 1:800, and then added to the sample hole of the test strip. The results show that Figure 12 ), the test strip can still detect positive when the serum is diluted to 1:200, and show negative when the dilution is higher than that.

[0049] Example 6: Application of the developed test strip in the diagnosis of nervous necrosis virus disease

[0050] The titer of the test strip is 10 4 TCID 50NNV at a concentration of 1.0×105 / ml was used to infect 3-month-old Epinephelus akaara with a body length of 7-10 cm. The experimental period lasted for five weeks, during which the behavior, pathological changes, and mortality of the experimental fish were observed and recorded. Blood samples were collected from 12 surviving Epinephelus akaara at 2, 3, 4, and 5 weeks after infection, and the serum was separated and detected using test strips and ELISA. After infection, the number of sick individuals showed an increasing trend from 2 to 5 weeks, and the cumulative mortality of Epinephelus akaara gradually increased from 3.3% to 20.0%, with the death peak mainly concentrated in the 3rd to 5th week after infection. The number of positive individuals detected by test strips was 10, 10, 11, and 11 at 2, 3, 4, and 5 weeks, respectively. ELISA results showed that all 48 serum samples were positive. A total of 56 serum samples were collected from Epinephelus akaara in a fish farm within one month, and the test strip detection results were all negative, which was consistent with the ELISA results. Combined with the test strip detection results and clinical manifestations, the diagnosis results can be divided into four categories: positive individuals with symptoms, which need to be immediately isolated; positive individuals without symptoms, which have a risk of transmission and need to be continuously monitored; negative individuals with symptoms, which need to be rechecked combined with other methods; and negative individuals without symptoms, which are healthy individuals and need to be regularly rechecked.

[0051] The above results show that the rabbit anti-Epinephelus akaara IgM polyclonal antibody is a gold label antibody, and the NNV capsid protein recombinant protein is marked on the detection line to develop a serological diagnostic test strip for Epinephelus akaara nervous necrosis virus disease. The test strip has good specificity, sensitivity, and stability, and can realize early and real-time diagnosis of the disease. The test strip has the advantages of not requiring professional instruments and equipment, on-site detection, simple operation, etc., and is suitable for on-site detection, providing strong technical support for the prevention and control of the disease.

Claims

1. A NNV capsid protein neutralizing epitope polypeptide, characterized in that The amino acid sequence of the neutralizing epitope polypeptide is SEQ ID NO:

1.

2. The neutralizing epitope polypeptide according to claim 1, wherein The amino acid sequence of the neutralizing epitope polypeptide is SEQ ID NO:

2.

3. Use of the neutralizing epitope polypeptide according to claim 1 or 2 in preparing antibodies.

4. The use according to claim 3, characterized in that The antibody is a polyclonal antibody.

5. Use of the neutralizing epitope polypeptide according to claim 1 or 2 as a detection antigen in the preparation of a product for detecting grouper NNV-specific antibodies.

6. The use according to claim 5, characterized in that The product is a serological diagnostic test strip for grouper nervous necrosis virus disease.

7. A serological diagnostic test strip for grouper nervous necrosis virus disease, characterized in that: The detection line of the test strip uses the neutralizing epitope polypeptide according to claim 1 or 2.

8. The diagnostic test strip according to claim 7, wherein The neutralizing epitope polypeptide is a mixed polypeptide of the neutralizing epitope polypeptides according to claims 1 and 2.

9. The diagnostic test strip according to claim 7, wherein: The gold-labeled antibody on the serological test strip is a rabbit anti-grouper IgM antibody.

10. The diagnostic test strip according to claim 7, wherein: The quality control line of the test strip is coated with goat anti-rabbit IgG secondary antibody.

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