Hybridoma cell line secreting monoclonal antibody against VP2 of infectious pancreatic necrosis virus, monoclonal antibody and application

Abstract

The present invention discloses a hybridoma cell line, monoclonal antibody and application that secretes anti-infectious pancreatic necrosis virus VP2 monoclonal antibody, which belongs to the field of bioengineering. The hybridoma cell line IPNV9-VP2 was deposited on August 17, 2022 at the China Center for Type Culture Collection, Wuhan University, Wuhan City, Hubei Province, with a deposit number of CCTCC C2022216. It is derived from the recombinant protein of infectious pancreatic necrosis virus VP2. The secreted anti-IPNV-VP2 monoclonal antibody has high purity and can specifically recognize IPNV-VP2 protein. It is used for ELISA to specifically detect the concentration of IPNV-VP2 protein in diseased fish tissues or the distribution of IPNV-VP2 in frozen tissue sections with high sensitivity and accuracy, providing parameter indicators for detecting viral infection. By monitoring the health status of salmon and trout in real time, it provides technical support for the sustainable development of salmon and trout aquaculture.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to a hybridoma cell line secreting an anti-infectious pancreatic necrosis virus VP2 monoclonal antibody, a monoclonal antibody and applications. Background Art

[0002] Infectious pancreatic necrosis is a highly contagious and acute viral disease of salmonids caused by the infectious pancreatic necrosis virus (IPNV). IPNV belongs to the genus Birnavirus and is the smallest RNA virus among known fish viruses. Its main host range and sensitive fish species include American trout, rainbow trout, grayling, Clark's salmon, coho salmon, calico salmon, lake trout, Atlantic salmon, Chinook salmon, etc. It mainly harms fry and fingerlings aged 14-70 days. The mortality rate can be as high as 80%-100% when the water temperature is between 10-12℃. The smaller the fish, the higher the mortality rate. Typical symptoms of diseased fish include abnormal swimming, dark body color, abdominal distension, bulging eyes, and hemorrhage and necrosis of internal organs such as the subcutaneous tissue and liver and pancreas. Highly pathogenic virus strains have a high mortality rate for two-month-old juvenile fish. Fish that survive the infection carry the virus for life and excrete pathogens through feces and sperm and eggs, becoming potential sources of infection. Due to the wide prevalence of the disease and high morbidity and mortality rates, it has caused significant economic losses to salmon and trout farming industries around the world.

[0003] The VP2 protein is the primary structural protein on the surface of the IPNV virus. It contains viral antigenic determinants and can induce the production of neutralizing antibodies, playing a key role in viral invasion. It is widely used in research on the detection and prevention of IPNV. Traditionally, the diagnosis of IPNV is based on epidemiology and primary symptoms. However, because IHNV and some other viruses can also cause subcutaneous hemorrhage and hemorrhagic necrosis of internal organs, these can be difficult to distinguish epidemiologically and symptomatically. Therefore, definitive diagnosis requires virus isolation and serological testing. Since Wolf first reported infectious pancreatic necrosis in juvenile salmon and trout in 1960, extensive research has been conducted worldwide on IPNV diagnostic techniques. Methods such as virus isolation (IV), nucleic acid detection (NAT), fluorescent antibody assay (FAT), virus neutralization test (VN), enzyme-linked immunosorbent assay (ELISA), and polymerase chain reaction (PCR) have been established. While IPNV can be initially diagnosed based on epidemiology, primary symptoms, and pathological changes, laboratory confirmation is still necessary to distinguish it from viral hemorrhagic septicemias caused by IHNV and other viruses.

[0004] Rapid and accurate disease diagnosis is crucial for the prevention and control of infectious pancreatic necrosis (IPN), yet there are currently no proven effective prevention and treatment methods. Isolating and identifying the suspected virus is the most straightforward diagnostic method, but virus isolation requires specific conditions and considerable time, limiting its widespread application. Currently, the most commonly used rapid diagnostic methods for IPN include serological tests, fluorescent antibody tests, and enzyme-linked immunosorbent assays (ELISAs). However, there are no reports on the identification of hybridoma cell lines specifically secreting anti-IPN VP2 monoclonal antibodies using recombinant proteins and their use in the production of monoclonal antibodies. Summary of the Invention

[0005] The first object of the present invention is to provide a hybridoma cell line that secretes anti-infectious pancreatic necrosis virus VP2 monoclonal antibodies, named IPNV9-VP2 (Infectious pancreatic necrosis virus VP2), which was deposited on August 17, 2022 in the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCC C2022216.

[0006] Preferably, the hybridoma cell line secreting anti-infectious pancreatic necrosis virus VP2 monoclonal antibody is derived from IPNV-VP2 recombinant protein.

[0007] The second object of the present invention is to provide the use of the hybridoma cell line secreting anti-IPN VP2 monoclonal antibody in the preparation of a reagent for diagnosing or detecting IPN infection.

[0008] The third object of the present invention is to provide an anti-infectious pancreatic necrosis virus VP2 monoclonal antibody, which is obtained by secretion of the hybridoma cell line that secretes the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody.

[0009] Preferably, the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody includes FITC- and CY5-labeled anti-IPNV-VP2 monoclonal antibody with high purity.

[0010] The fourth object of the present invention is to provide the use of the above-mentioned anti-IPN VP2 monoclonal antibody in the preparation of a reagent for diagnosing or detecting IPN infection.

[0011] The fifth object of the present invention is to provide an infectious pancreatic necrosis virus diagnostic kit, which comprises the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody.

[0012] Preferably, the infectious pancreatic necrosis virus diagnostic kit includes a box body and a reagent strip placed in the box body, wherein: the reagent strip includes a bottom plate and a sample pad, a conjugation pad, a nitrocellulose membrane and a water-absorbing pad overlapped end to end along the length of the bottom plate; the conjugation pad is coated with the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody labeled with colloidal gold; the nitrocellulose membrane is respectively coated with the anti-IPNV-VP2 VP2 protein detection line N line and the quality control line C line coated with sheep anti-mouse IgG antibody.

[0013] The sixth object of the present invention is to provide the use of the above-mentioned infectious pancreatic necrosis virus diagnostic kit in the rapid detection of salmon and trout infectious pancreatic necrosis virus antigens.

[0014] The present invention obtains IPNV-VP2 recombinant protein through prokaryotic expression in Escherichia coli, and uses it for preparing anti-IPNV-VP2 monoclonal antibodies and identifying them after purification, laying the foundation for studying the infection mechanism of IPNV and developing a rapid diagnostic kit for the virus, and also providing technical support for the prevention and control of fish diseases.

[0015] In some embodiments, the method for preparing the hybridoma cell line capable of secreting anti-IPNV-VP2 monoclonal antibodies is as follows:

[0016] Immunization of mice: Four SPF BALB / c female mice were selected and immunized four times with a 14-day immunization cycle. For the first immunization, 60 μg of IPNV-VP2 recombinant protein was injected subcutaneously. For the booster immunization, 30 μg was injected per mouse each time. Ten days after the last immunization, the mice were anesthetized and blood was collected from their orbits. The titer of the immunized mice was determined by ELISA.

[0017] Cell fusion: Before fusion, mice were immunized by intraperitoneal injection of 50 μg IPNV-VP2 recombinant protein. Mouse spleen cells were taken and fused with SP2 / 0 cells using the PEG method. The fused cells were transferred to a semi-solid culture medium (containing HAT) for culture.

[0018] Screening of positive hybridoma cells: Monoclones grown in semi-solid culture medium are picked and cultured in 96-well culture plates. The selected clones are screened twice and subclassified using the ELISA method to obtain IgG type positive hybridoma cell lines, which are then cultured in large quantities and frozen.

[0019] In some embodiments, the hybridoma cell line secreting anti-IPNV-VP2 monoclonal antibodies is injected into mice, and then mouse ascites is collected to prepare anti-IPNV-VP2 monoclonal antibodies. Two highly pure (>90%) anti-IPNV-VP2 monoclonal antibodies are obtained through affinity purification using a HiTrap rProtein A FF column. ELISA assay shows an antibody affinity constant of 9.60E+09, and Western blot analysis reveals that both antibodies specifically recognize the eukaryotic IPNV-VP2 protein without cross-reacting with other proteins. The anti-IPNV-VP2 monoclonal antibodies are labeled with FITC and CY5 fluorescent dyes, respectively. The FITC- and CY5-labeled anti-IPNV-VP2 monoclonal antibodies produced have high potency and specificity and can be used to specifically detect IPNV-VP2 protein concentrations in diseased fish using ELISA. Furthermore, laser confocal microscopy is used to observe the distribution of IPNV-VP2 in frozen tissue sections, providing a parameter indicator for detecting viral infection and enabling real-time monitoring of the health of salmon and trout.

[0020] Compared with the prior art, the beneficial effects of the present invention are: in response to the current problem of lack of anti-IPNV-VP2 monoclonal antibodies, IPNV-VP2 recombinant protein is used as an antigen to immunize mice and then screen to obtain a positive hybridoma cell line that specifically secretes anti-IPNV-VP2 monoclonal antibodies of IgG type. The prepared anti-IPNV-VP2 monoclonal antibodies have high purity (>90%) and an affinity constant of 9.60E+09. They can specifically recognize IPNV-VP2 protein and have high detection sensitivity and accuracy. They can be used for ELISA to specifically detect the IPNV-VP2 concentration in diseased fish tissues, and the distribution of IPNV-VP2 in different tissues is detected by frozen tissue sections, providing parameter indicators for detecting viral infection, and can monitor the health status of salmon and trout in real time, providing technical support for the sustainable development of salmon and trout farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the purification of IPNV-VP2 recombinant protein and SDS-PAGE protein electrophoresis verification in Example 1.

[0022] Figure 2 It is the antibody immune titer in Example 2.

[0023] Figure 3 The purity of the antibody in Example 3.

[0024] Figure 4 is the affinity constant of the antibody in Example 3.

[0025] Figure 5The Western blot test for antibody specificity in Example 4 showed that the IPNV-VP2 eukaryotic expression protein reacted specifically with both the monoclonal antibody positive serum and 9#. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and specific embodiments of the present invention, but the protection scope of the present invention is not limited to the following.

[0027] Example 1: Preparation of IPNV-VP2 antigen

[0028] Construction of prokaryotic expression plasmid of IPNV-VP2

[0029] The VP2 prokaryotic expression plasmid was constructed by Jinweizhi Biotechnology Co., Ltd. The IPNV-VP2 sequence was obtained from the NCBI GenBank (GenBank: sp|Q703G9.1|). The expression vector pET-21d was purchased from Jinweizhi Biotechnology Co., Ltd. and maintained in our laboratory. The mature peptide DNA sequence of IPNV-VP2 was optimized (E. coli) and the gene pET21d-IPNV-VP2 was synthesized conventionally. The 5' (NcoI) and 5' UTR ([CT]) and 3' (BamHI) residues were added. The gene was then cloned into the pET21d (Ampicillin) vector using the 5' NcoI and 3' BamHI residues to prepare mini-scale recombinant plasmid DNA. pET21d-IPNV-VP2 was transformed into competent expression strains Rosetta, C43, BL21, and DE3 cells. Single colonies were selected by plating and PCR screening.

[0030] Inducible expression and purification of IPNV-VP2 recombinant protein

[0031] The positive clone strains were inoculated into 10 mL of LB liquid medium (containing 100 μg / mL ampicillin) at a ratio of 1:100 and cultured in a 37°C incubator at 170 rpm to an OD value of 0.4-0.6 (in duplicate). The first 10 mL of bacterial culture was used as a negative control, while the other was supplemented with 10 μL of 1 mol / mL IPTG and cultured with shaking for 12 hours to induce protein expression. Then, 1 mL of bacterial culture was transferred to a 1.5 mL EP tube and centrifuged at 12,000 rpm / min for 2 minutes. The supernatant was discarded, and 40 μL of sterile PBS buffer and 10 μL of 5× SDS protein loading buffer were added. The mixture was then heated in a metal bath at 100°C for 10 minutes. After the samples were cooked, they were centrifuged and subjected to SDS-PAGE gel electrophoresis to analyze the expression of the target protein. If expression is performed, a large amount of protein is induced. After centrifugation, the induced bacteria are discarded and washed twice with sterile PBS. Then, they are broken with an ATS micro-ultrahigh pressure homogenizer, and the supernatant and inclusion bodies are collected. SDS-PAGE electrophoresis analysis is performed. The recombinant protein exists in the form of inclusion bodies. Then, dilution and renaturation, nitrogen pressure concentration and AKTA protein purification instrument are performed. The obtained protein is measured by BCA protein concentration kit and stored at -80℃ for later use. Figure 1 ).

[0032] Example 2: Preparation of hybridoma cell lines

[0033] (1) Experimental equipment and reagents: Sterile surgical instruments: three pairs of scissors, three pairs of forceps, a cell strainer, a syringe core, a petri dish, two 500 mL beakers, two 50 mL centrifuge tubes, three 15 mL centrifuge tubes; IMDM medium, IMDM complete medium (containing 15% serum), 2.2% methylcellulose (SIGMA, M0262-100G), 10 mL newborn calf serum, PEG1500 (Roche, 78364), HAT ( Sigma, H0262-10VL), HT (Sigma, H0137-10VL), coating solution (sodium carbonate-sodium bicarbonate buffer, pH 9.6), pH 7.4 PBS buffer, blocking solution (2% milk), washing solution: PBS-T (0.05% Tween, PBS), color development solution: 1% solution A + 10% solution B (solution A: 1% TMB; solution B: 0.1% H2O2), stop solution: 2 M sulfuric acid, secondary antibody: goat anti-mouse IgG / HRP.

[0034] (2) Immunization of mice: Four SPFBALB / c female mice, numbered 1, 2, 3, and 4, were subcutaneously immunized with 60 μg of IPNV-VP2 recombinant protein per mouse. The mice were then given three booster immunizations at a dose of 30 μg per mouse, with two weeks between each immunization. Blood was collected from the eye sockets.

[0035] (3) ELISA to detect antibody titer: 2 μg / mL IPNV-VP2 recombinant protein was used for coating overnight at 4°C; the cells were washed three times with washing solution and blocked with blocking solution at 37°C for 1 hour; the monoclonal antibody was diluted 2-fold starting from 200 times, the blank control was PBS, and the negative control was a 200-fold dilution of negative serum. The diluted antibody was added to the protein-coated wells, and three replicates were set for each concentration gradient. The cells were incubated at 37°C for 2 hours, then washed three times and patted dry; the HRP-labeled goat anti-mouse secondary antibody was diluted 1:10000, mixed, and 100 μL was added to each well. The cells were incubated at 37°C for 1 hour, then washed three times and patted dry; 100 μL of TMB single-component colorimetric solution was then added to each well, and the cells were developed at 37°C with the light blocked to the desired depth (10–30 min); finally, 50 μL of stop solution (2 mM H2SO4) was added to each well, and the OD value at 450 nm was measured using an enzyme-labeled instrument. The results are shown in the following table. Figure 2 As shown, the antibody titer of mouse #1 was greater than 25600, and mouse #1 was selected for intraperitoneal injection of 50 μg immunogen for cell fusion experiment.

[0036] (4) Cell fusion experiment: Sp2 / 0 cells in good condition were gently blown off the wall of the culture flask and sucked into a 50 mL centrifuge tube. Anesthetize the mouse and remove the eyeball to collect blood, then kill by cervical dislocation and soak in 75% alcohol for 5 minutes. Pour a small amount of serum-free IMDM into the dish, and place the cell sieve and syringe inner core into the dish. Use scissors and forceps to remove the mouse spleen and place it on the cell sieve. Use the inner core of the syringe to gently crush the spleen, and suck the crushed cells into the centrifuge tube containing sp2 / 0, and centrifuge at 1500 rad / min for 5 minutes. Use scissors and forceps to remove the mouse thymus and crush it. The crushed thymocytes were transferred to a 15 mL centrifuge tube, and 2 mL of HAT and 2 mL of HT were added and placed in the incubator for use. After the centrifugation of the cells, the supernatant was discarded, and the cells were carefully and gently blown evenly with serum-free IMDM and centrifuged (1500 rad / min, 5 minutes). Discard the supernatant of the centrifuged cells, tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, slowly add 1mL of PEG within 1 minute, and let it stand in warm water for 1 minute. Then, slowly add 2mL of serum-free IMDM within 2 minutes, followed by 8mL of serum-free IMDM within 2 minutes. Centrifuge at 1000rad / min for 5 minutes. Discard the supernatant, add 10mL of serum, carefully blow the cells evenly, and pour in the thymocytes prepared earlier. Then add 25mL of sterile semi-solid culture medium, mix thoroughly, and then evenly pour into 30 cell culture dishes. Place the cell culture dishes in a humidified box and culture in an incubator.

[0037] (5) Monoclonal cell screening: First, 10 plates × 93 cell clones were selected and cultured in 96-well cell culture plates (previously plated with thymocytes, 100 μL / well). The selected clones were then screened using the ELISA method. IPNV-VP2 protein was diluted to 2 μg / mL with coating solution, 100 μL / well, at 4°C overnight, and then washed three times with washing solution. Block with 2% milk blocking buffer (200 μL / well) and incubate at 37°C for 2 hours. Wash three times with washing buffer. Add 100 μL / well of primary antibody (cell culture supernatant), negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000-fold in PBS) and incubate at 37°C for 1 hour. Wash three times with washing buffer. Add 100 μL / well of secondary antibody diluted 20,000-fold in PBS and incubate at 37°C for 1 hour. Remove and wash three times with washing buffer. Develop the color with 100 μL / well of colorimetric solution for approximately 5 minutes. Add 50 μL of stop solution to each well. Measure absorbance at dual wavelengths (450 and 630 nm) and record the data. Perform the second screening procedure as above.

[0038] (6) Antibody subclass identification: Dilute the coated antibody to 0.5 μg / mL with 100 mM PBS (pH 7.4), add 0.1 mL to each well, and incubate at 4°C overnight. Wash twice with PBS-T; add 200 μL of blocking solution to each well and incubate at 37°C for 2 h. Wash three times with PBS-T; add 100 μL of hybridoma supernatant to each well and incubate at 37°C for 1 h. Wash three times with PBS-T; add 0.1 mL of HRP-labeled antibody diluted 1:1000 or 1:2000 (goat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA) with blocking solution to the appropriate wells and incubate at 37°C for 1 h. Wash three times with PBS-T; add 50 μL of substrate solution to each well, measure the absorbance at dual wavelengths (450, 630) within 10–20 min, and record and save the data (Table 1). Finally, five IgG-positive hybridoma cell lines were obtained, cultured in large quantities, and then frozen.

[0039] Table 1: Monoclonal cell line subtype identification and related information

[0040]

[0041] Example 3: Preparation of anti-IPNV-VP2 monoclonal antibodies

[0042] The hybridoma cell lines screened above were cultured at a rate of 5×10 7The dose of 10 cells was intraperitoneally injected into BALB / c mice. After about two weeks, when the abdomen of the mice was obviously swollen, the ascites was collected to obtain anti-IPNV-VP2 monoclonal antibodies. The antibodies were then affinity purified using HiTrap rProtein A FF columns and the purity of the antibodies was determined by SDS-PAGE. Figure 3 As shown, the antibody purity was high and greater than 90%.

[0043] The affinity constant of the purified antibody was then detected by ELISA, that is, 2 μg / mL IPNV-VP2 recombinant protein was used for coating overnight at 4°C; 2% milk was used for blocking at 37°C for 2 h; the antibody was serially diluted from 200 times, the blank control was PBS, both at 100 μL / well, and incubated at 37°C for 1 h; washed three times with washing solution, and the secondary antibody diluted 20,000 times with PBS was added at 100 μL / well, and incubated at 37°C for 1 h; after removal, washed three times with washing solution, and color was developed with 100 μL / well of color development solution for about 5 min, and 50 μL of stop solution was added to each well to stop the color development; the absorbance value was measured, and the data was recorded and saved. The affinity constant of the monoclonal antibody was 9.60E+09 ( Figure 4 ).

[0044] A portion of the anti-IPNV-VP2 monoclonal antibody was labeled with FITC and CY5 fluorescent dyes. Prior to labeling, the anti-IPNV-VP2 monoclonal antibody sample was dialyzed three or more times against 0.1 mol / L carbonate buffer, pH 9.0, at 4°C. The dialyzed antibody was then transferred to a brown bottle, and 75 μL of 1 mg / mL FITC and CY5 fluorescent dye was added to 1 mg of antibody with slow stirring. The mixture was incubated at 4°C overnight. The reacted anti-IPNV-VP2 monoclonal antibody was dialyzed four or more times against 0.01 mol / L PBS buffer, pH 7.4, at 4°C until the dialyzate was colorless and stored at -20°C until needed.

[0045] Example 4: Specificity Analysis of IPNV-VP2 Monoclonal Antibodies

[0046] (1) Western blotting to detect eukaryotic protein expression

[0047] HEK293T cells were transfected with the pCDNA3.1-IPNV-VP2-Myc plasmid, and cells transfected with the pCDNA3.1-empty plasmid served as a control group. Cells were harvested 24 hours after transfection and protein extracts were used for Western blotting. Protein samples were transferred to a PVDF membrane using electroporation (25 V, 25 mA, 7 min). The membrane was blocked with 5% skim milk at 4°C for 2 h and then incubated with unlabeled IPNV-VP2 monoclonal antibody (1:200 dilution, overnight at 4°C). HRP-conjugated goat anti-mouse secondary antibody (1:10,000 dilution) was incubated at 4°C for 1 h. The membrane was then washed three times with TBS-T buffer (5 min each). The membrane was then imaged and analyzed using an Odyssey CLX Imaging System. The results showed that when the Myc tag antibody carried by the plasmid was incubated, the corresponding target bands appeared in the cell lysate samples transfected with pCDNA3.1-IPNV-VP2-Myc, proving that IPNV-VP2 was well expressed in HEK293T cells. No bands appeared in the protein samples transfected with the empty plasmid. The screened IPNV9-VP2 monoclonal antibodies were able to specifically recognize the IPNV-VP2 protein expressed in HEK293T cells, and the incubated bands were consistent with the theoretical molecular weight, which was consistent with the results of Myc antibody incubation. The IPNV-VP2 eukaryotic protein may be modified during the expression process, resulting in the appearance of multiple bands ( Figure 5 ).

[0048] Example 5: Preparation and application of a rapid detection kit for IPNV virus antigen

[0049] (1) Preparation of sample pad: The sample pad uses SB-08 glass fiber membrane and is prepared with sample pad treatment solution, which contains PBS buffer, casein and Tween-20; the PBS buffer uses PBS buffer salt with a concentration of 0.01–0.1 M and a pH of 7.4; the casein uses casein with a mass fraction concentration of 0.1%–1%; the Tween-20 uses Tween-20 with a volume fraction concentration of 0.1%–1%; in this embodiment, the sample pad treatment solution comprises 0.01 M PBS buffer with a pH of 7.4, 1% casein by mass and 1% Tween-20 by volume; the sample pad is immersed in the sample pad treatment solution for 30 min, and the excess liquid is discarded. The sample pad after soaking is placed in a dry environment at a temperature of 37°C to dry for 12 h, taken out and set aside.

[0050] (2) Preparation of conjugate pad: The conjugate pad uses SB-08 glass fiber membrane; first, prepare the gold-labeled antibody, take 1 mL of colloidal gold solution, the wavelength corresponding to the maximum visible light absorbance value of the colloidal gold solution is 520–530 nm, and add 10 μL of potassium carbonate with a concentration of 0.02–0.5 M, and mix for 2 minutes; add anti-IPNV-VP2 protein monoclonal antibody with a final concentration of 5-20 μg / mL as the labeled antibody to the mixed solution, and mix for 10 minutes; then add 0.1 mL BSA was used as a blocking agent at a concentration of 1%–10% by mass, and the mixture was mixed for 30 min. The solution after adding BSA was centrifuged at 4°C and 12500 rpm / min for 10 min, the supernatant was carefully discarded, and the precipitate was resuspended with 40 μL of Tris-HCl buffer at a pH of 8.0. The concentration of Tris-HCl buffer was 0.01–0.1 M. Sucrose was added to the resuspended solution as a protective agent, and the final concentration of sucrose was 5%–30%. The mixture was thoroughly mixed to prepare the product. Gold-labeled antibody; the wavelength corresponding to the maximum visible light absorbance value of the colloidal gold solution in the present invention is 525 nm, the potassium carbonate concentration is 0.2 M, the final concentration of the anti-IPNV-VP2 protein monoclonal antibody is 5 μg / mL, the BSA concentration is 10%, the Tris-HCl buffer concentration is 0.01 M, and the final sucrose concentration is 20%; then, the prepared gold-labeled antibody is sprayed on the conjugate pad, dried in a dry environment at a temperature of 37° C. for 5 hours to solidify the gold-labeled antibody on the conjugate pad, and sealed for storage.

[0051] (3) Membrane spotting: Prepare membrane coating solution; the membrane coating solution includes PBS buffer salt, isopropanol, Triton X-100 and trehalose; the PBS buffer salt uses a final concentration of 0.001–0.05M and pH = 8.2; the isopropanol uses an isopropanol with a final volume concentration of 1%–5%; the Triton X-100 uses a final volume concentration of 0.0001%–0.001%; the trehalose uses a final concentration of 0.1%–1%; the final concentration of PBS buffer salt is 0.01M, the final concentration of isopropanol is 2%, and the final concentration of Triton The final concentration of X-100 was 0.0005%, the final concentration of trehalose was 0.3%, and the dilution concentration of the anti-IPNV-VP2 protein monoclonal antibody was 2 mg / mL. The anti-IPNV-VP2 protein monoclonal antibody was diluted to 1–2 mg / mL using the streaking coating solution. The diluted antibody was then streaked on the nitrocellulose membrane adjacent to the binding pad at one end as the test line, and the goat anti-mouse IgG antibody was streaked on the other end of the nitrocellulose membrane as the quality control line. The streaked nitrocellulose membrane was placed in a dry environment at 37°C for 48 hours to obtain a nitrocellulose membrane with a test line and a quality control line.

[0052] (4) Assembly of the reagent card: The sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad processed in steps 1 to 3 are sequentially bonded to the base plate and cut into 3.00 mm wide reagent strips, which are then placed in a box template to form a test kit. The box is provided with a sample loading hole and an observation window. The sample loading hole is located above the sample pad, and the observation window is located above the test line and the quality control line.

[0053] (5) Prepare the test kit as follows: dilute the sample to be tested with a sample diluent, drip the diluted sample to be tested onto the sample pad of the reagent strip through the sample injection hole, and observe the test line and quality control line results on the reagent strip through the observation window within 15–20 minutes.

[0054] The results interpretation on the reagent strip include: ① Positive result: When a red stripe appears on the test line N and a red stripe appears normally on the quality control line C, the test result is judged to be positive; ② Negative result: If only a red stripe appears on the quality control line C and the test line N does not show any color, the test result is judged to be negative; ③ Invalid result: When a red stripe does not appear on the quality control line C, the test result is invalid.

[0055] The anti-IPNV-VP2 monoclonal antibodies obtained by the above examples have the following application prospects:

[0056] ① The application of monoclonal antibodies to quantitative analysis of IPNV protein in host cells.

[0057] ②The application of monoclonal antibodies to analyze the localization of IPNV protein in host tissues.

[0058] ③Application of monoclonal antibody Western blotting.

[0059] Among them, the IPNV virus rapid detection kit is used to quickly detect diseased fish, and the specific operation of ELISA to detect the content of IPNV-VP2 protein is as follows:

[0060] (1) Antigen coating: IPNV-VP2 was used as the antigen to be tested. IPNV-VP2 recombinant protein was used as the standard and diluted to different concentrations with coating solution to prepare a standard curve. 100 μL was added to each well of the ELISA plate. 2 μg / mL BSA was set as a negative control and the plate was incubated at 4°C overnight.

[0061] (2) Washing: Discard the coating solution, wash three times with washing solution, 3 min each time, 500 μL / well, and pat dry.

[0062] (3) Blocking: Add 200 μL of blocking solution to each well, incubate at 37°C for 2 h, then wash three times and pat dry.

[0063] (4) Primary antibody incubation: Dilute the prepared IPNV-VP2 monoclonal antibody at 1:1000 with antibody diluent, add 100 μL to each well, and incubate the same sample three times at 37°C for 2 h, then wash three times and pat dry.

[0064] (5) Secondary antibody incubation: HRP-labeled goat anti-mouse secondary antibody was diluted 1:10,000, mixed and added to each well with 100 μL, incubated at 37°C for 1 h, then washed 3 times and patted dry.

[0065] (6) Color development: Add 100 μL of TMB single-component color development solution to each well and develop the color at 37°C in the dark until the desired color depth (10-30 min).

[0066] (7) Termination reaction: Add 50 μL of stop solution (2 mM H2SO4) to each well and measure the OD value at 450 nm using a microplate reader.

[0067] 2. Immunohistochemistry (laser confocal microscopy) analysis

[0068] (1) Fixation and embedding of tissues of diseased fish infected with IPNV: (Fixation) Immediately after sampling the tissue to be tested, place it in 4% PFA tissue fixative and fix it at 4°C overnight; (Dehydration) Place the tissue sample in 20% sucrose solution for dehydration until it sinks, then replace it with 30% sucrose solution for dehydration. After sinking, replace it with a new 30% sucrose solution until the tissue sinks to the bottom, and let it stand at room temperature for 1 hour; (Embedding) Fold tin foil into small pieces, pour the embedding agent OCT into it, position the tissue, place it in -80°C, and wait for it to solidify before slicing.

[0069] (2) Preparation of frozen sections: The frozen tissue was quickly placed in a cryostat that was precooled to -20°C. When the embedding agent was completely solidified, the tissue embedding block was fixed to the knife holder and the tissue embedding block was trimmed to a smooth surface with a single-sided blade. The slices were then trimmed (80 μm) and the thickness of the slices was adjusted to 6 μm. The tissue was adhered to a slide and marked.

[0070] (3) Immunofluorescence staining: Remove the slides from -80°C and reheat for about 30 minutes until the slides are dry. Place the slides in 0.01M PBS buffer for 5 minutes (on a shaker), then change to 0.3% Triton X-100 permeabilization for 15 minutes (on a shaker), wash with PBS three times, 5 minutes each time (on a shaker), place the slides in sodium citrate solution, and heat in a microwave oven to about 98°C (beginning to bubble) for antigen retrieval. Stop for 5 minutes, then reheat, repeat three times for a total of 15 minutes, and then take out and cool to room temperature for about 1 hour. Wash with PBS three times, 10 minutes each time (shaking), use filter paper to absorb the liquid around the front and back tissues, circle the tissue with a histochemical pen, place the slide in a humidified box, add the serum from the primary antibody for blocking for 30 minutes (shaking), shake off the serum, add about 35 μL / slide of appropriately diluted FITC or CY5-labeled IPNV-VP2 monoclonal antibody, place in a humidified box at 4°C overnight, remove the humidified box from 4°C, rewarm for about 30 minutes, use a pipette to remove the excess liquid (primary antibody) on the slide, place in PBS in the dark for 3 times, 10 minutes each time (shaking), add about 30 μL of DAPI (depending on the size of the tissue), and stain in the dark for 3-5 minutes. Place in PBS in the dark for 3 times, 5 minutes each time (shaking), add fluorescence signal enhancement mounting medium to seal the slide, place in a humidified box at 4°C for 1 hour, and finally observe and photograph using a laser confocal microscope.

[0071] The above description of the embodiments is intended to facilitate understanding and application of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments, and improvements and modifications that do not depart from the scope of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. The hybridoma cell line secreting anti-infectious pancreatic necrosis virus VP2 monoclonal antibody, named IPNV9-VP2 (Infectious pancreatic necrosis virus VP2), was deposited on August 17, 2022, in the China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the deposit number CCTCCC2022216.

2. Use of the hybridoma cell line secreting anti-IPN VP2 monoclonal antibody according to claim 1 in the preparation of a reagent for diagnosing or detecting IPN infection.

3. An anti-infectious pancreatic necrosis virus VP2 monoclonal antibody, characterized in that: The antibody is obtained by secreting the hybridoma cell line secreting the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody as claimed in claim 1.

4. Use of the anti-IPN VP2 monoclonal antibody according to claim 3 in the preparation of a reagent for diagnosing or detecting IPN infection.

5. A diagnostic kit for infectious pancreatic necrosis virus, characterized in that: It comprises the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody according to claim 3.

6. The infectious pancreatic necrosis virus diagnostic kit according to claim 5, characterized in that: The infectious pancreatic necrosis virus diagnostic kit comprises a box body and a reagent strip placed in the box body, wherein: The reagent strip comprises a bottom plate and a sample pad, a conjugate pad, a nitrocellulose membrane and a water-absorbing pad which are sequentially overlapped end to end along the length direction of the bottom plate; The conjugate pad is coated with the anti-infectious pancreatic necrosis virus VP2 monoclonal antibody according to claim 3 labeled with colloidal gold; The nitrocellulose membrane is coated with a VP2 protein detection line N line coated with an anti-IPNV-VP2 and a quality control line C line coated with a goat anti-mouse IgG antibody.

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