Hybridoma cell strain secreting GCRV VP4 protein monoclonal antibody and monoclonal antibody and application thereof

Abstract

The invention provides a hybridoma cell strain secreting a grass carp reovirus VP4 protein monoclonal antibody as well as the monoclonal antibody and application thereof, and belongs to the technical field of aquatic reovirus detection. The hybridoma cell strain is named as GCRV-VP4-6E7B12, and is preserved in the China Center for Type Culture Collection on December 10, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2024160. The grass carp reovirus VP4 protein monoclonal antibody 6E7B12 secreted by the hybridoma cell strain GCRV-VP4-6E7B12 has the advantage of being specifically combined with an antigen. The enzyme-labeled monoclonal antibody is used as a detection antibody, the VP4 protein polyclonal antibody is used as a capture antibody, the content of the grass carp reovirus VP4 protein in a sample to be detected can be quantitatively detected by adopting a double-antibody sandwich ELISA method, the detection range is 1-32 ng / mL, and the lower detection limit is 1 ng / mL. The detection method has the advantages of high sensitivity and wide detection range.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic reovirus detection, and specifically relates to a hybridoma cell strain secreting a monoclonal antibody against the VP4 protein of a grass carp reovirus (GCRV), the monoclonal antibody secreted by the hybridoma cell strain, and applications thereof. Background Art

[0002] Grass carp reovirus (GCRV) is a double-stranded RNA virus belonging to the genus Aquatic Reovirus of the family Reoviridae. The current grass carp reovirus strains prevalent in China are divided into three genotypes. GCRV-873 and GCRV-GZ1208 are representative strains of genotype I, GCRV-104 is a representative strain of genotype III, and most strains are genotype II. GCRV can cause severe hemorrhagic disease in grass carp in summer. When the water temperature is between 24℃ and 30℃, the mortality rate can be as high as 100%. In addition to infecting grass carp (Ctenopharyngodon idellus), GCRV has also been found to infect black carp (Mylopharyngodon piceus), wheatear fish (Pseudorasbora parva) and rare minnow (Gobiocyprisrarus), causing huge economic losses to my country's freshwater fish farming industry.

[0003] Existing methods for detecting grass carp reovirus include PCR-based nucleic acid detection methods (such as methods disclosed in CN106811550B, CN109971890A, CN108504783A, etc.), RT-RPA-based detection methods (such as methods disclosed in CN112680544B), loop-mediated isothermal nucleic acid amplification technology (such as methods disclosed in CN104673935A and CN104561379A), and RT-LAMP visualization detection methods (such as methods disclosed in CN104450961A, CN104450962A, and CN102703608A). In addition, there is an ELISA detection method based on IgM antibodies (CN102876809B), the principle of which is to detect whether grass carp serum samples contain GCRV-II antibodies. At present, there is no ELISA detection method and kit based on direct detection of genotype II grass carp reovirus. Summary of the invention

[0004] The first object of the present invention is to provide an immunogenic grass carp reovirus VP4 protein, and the nucleotide sequence encoding the grass carp reovirus VP4 protein is shown in SEQ ID No.1.

[0005] The second object of the present invention is to provide a hybridoma cell line that secretes a monoclonal antibody against the VP4 protein of grass carp reovirus. The hybridoma cell line is named GCRV-VP4-6E7B12 (Hybridoma cell line GCRV-VP4-6E7B12), which has been deposited in the China Center for Type Culture Collection on December 10, 2024, with the deposit address being Wuhan University, Wuhan City, Hubei Province, China, and the deposit number being CCTCC NO: C2024160. The hybridoma cell line secretes a monoclonal antibody against the VP4 protein of grass carp reovirus encoded by the sequence shown in SEQ ID No.1.

[0006] The third object of the present invention is to provide the use of the hybridoma cell line with the deposit number of CCTCC NO: C2024160 in the preparation of monoclonal antibodies against the VP4 protein of grass carp reovirus.

[0007] The fourth object of the present invention is to provide a monoclonal antibody 6E7B12 against the VP4 protein of grass carp reovirus secreted by a hybridoma cell line with a deposit number of CCTCC NO: C2024160. The monoclonal antibody 6E7B12 is identified as an IgG1 antibody, and its light chain is of κ subtype.

[0008] The fifth object of the present invention is to provide the use of the grass carp reovirus VP4 protein encoded by the sequence shown in SEQ ID No.1 or the hybridoma cell line with a deposit number of CCTCC NO: C2024160 or the monoclonal antibody 6E7B12 in the preparation of a product for detecting grass carp reovirus.

[0009] In a preferred embodiment, the product for detecting grass carp reovirus is a kit.

[0010] In a further preferred embodiment, the kit is a double antibody sandwich ELISA kit.

[0011] A sixth object of the present invention is to provide an ELISA kit for detecting grass carp reovirus, wherein the ELISA kit comprises the monoclonal antibody 6E7B12.

[0012] In a preferred embodiment, the ELISA kit further comprises a polyclonal antibody against the VP4 protein of grass carp reovirus as a capture antibody; and the monoclonal antibody 6E7B12 is used as a detection antibody after being labeled with an enzyme.

[0013] In a further preferred embodiment, the monoclonal antibody 6E7B12 is labeled with HRP (horseradish peroxidase) and used as a detection antibody.

[0014] In a further preferred embodiment, the ELISA kit further comprises a blocking solution, which is a 1×PBS buffer solution containing 0.1% (v / v) Tween-20, 0.5 wt % BSA and 0.04 wt % Block ACE.

[0015] In a further preferred embodiment, the ELISA kit further comprises grass carp reovirus VP4 protein as an antigen, and the coating concentration of the antigen is 1-32 ng / mL.

[0016] The technical solution of the present invention has the following beneficial effects: (1) The monoclonal antibody 6E7B12 against the VP4 protein of grass carp reovirus secreted by the hybridoma cell line GCRV-VP4-6E7B12 of the present invention has the advantages of strong specificity and high antigen binding ability.

[0017] (2) The ELISA kit constructed by using the enzyme-labeled grass carp reovirus VP4 protein monoclonal antibody provided by the present invention as the detection antibody and the grass carp reovirus VP4 protein polyclonal antibody as the capture antibody can quantitatively detect the content of grass carp reovirus VP4 protein in the sample to be tested, with a detection range of 1-32 ng / mL and a detection limit of 1 ng / mL. The kit has the advantages of high sensitivity and wide detection range. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram showing the SDS-PAGE electrophoresis results of various solutions in the process of purifying the VP4 protein when preparing the VP4 protein in the present invention; Figure 2 This is a diagram showing the SDS-PAGE electrophoresis result of the target protein collected after the VP4 protein is purified when the VP4 protein is prepared in the present invention; Figure 3 This is a diagram showing the SDS-PAGE electrophoresis result of the purified VP4 protein monoclonal antibody prepared in the present invention; Figure 4 This is a diagram showing the results of Western-Blot identification of the VP4 protein polyclonal antibody prepared in the present invention; Figure 5 The standard curve diagram is a double antibody sandwich ELISA detection of VP4 protein using the VP4 protein monoclonal antibody and VP4 protein polyclonal antibody prepared in the present invention.

[0019] Deposit description: The hybridoma cell line secreting the grass carp reovirus VP4 protein monoclonal antibody encoded by the nucleotide sequence shown in SEQ ID No. 1 in this application is named GCRV-VP4-6E7B12 (Hybridoma cell line GCRV-VP4-6E7B12), which was deposited in the China Center for Type Culture Collection on December 10, 2024, with the deposit address being Wuhan University, Wuhan City, Hubei Province, China, and the deposit number being CCTCC NO: C2024160. DETAILED DESCRIPTION

[0020] The following content is combined with the embodiments to clearly and completely describe the technical solution of the present application so that those skilled in the art can fully understand the present application. Obviously, the described embodiments are only some preferred embodiments of the present application, rather than all embodiments. Any equivalent transformation or substitution made to the following implementation modes by those of ordinary skill in the art without creative work belongs to the protection scope of the present application.

[0021] The sources or components of the raw materials such as culture medium, kits, plasmids, competent cells, etc. used in the following examples are as follows: The main components of LB liquid medium are: 10g / L tryptone, 5g / L yeast extract, and 10g / L sodium chloride; The main components of LB solid medium are: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 7.5g / L agar powder; 5× reducing loading buffer is SDS-PAGE protein loading buffer (5×) purchased from Shanghai Bio-Tech Biotechnology Co., Ltd., product number P0015; The main components of incomplete 1640 medium: 80% 1640 basal medium, 20% fetal bovine serum; The main components of HAT complete medium are: 80% 1640 basal medium, 20% fetal bovine serum, 1×HAT (Gibco™ 50×HAT, the supplement is a liquid mixture of sodium hypoxanthine (5mM), aminopterin (20µM) and thymidine (0.8mM)).

[0022] pET-28a vector was purchased from Wuhan Tiande Biotechnology Co., Ltd., with the catalog number TD-M028. Competent cells BL21 (DE3) were purchased from Wuhan Tiande Biotechnology Co., Ltd. BALB / c mice were purchased from Alvance Biotechnology Co., Ltd. SP2 / 0 cells were purchased from Wuhan Tiande Biotechnology Co., Ltd. HRP-goat anti-mouse IgG was purchased from Wuhan Tiande Biotechnology Co., Ltd. New Zealand white rabbits were purchased from Alvance Biotechnology Co., Ltd. Type II GCRV virus was obtained from diseased grass carp in the wild. The viral RNA was amplified by RT-PCR (forward primer: 5'-ATTCCACCACCTCCTGTGC-3'; reverse primer: 5'-CCACACCTCATCTGGCCATT-3'), Sanger sequencing and sequence alignment (the reference sequence is Grass carpreovirus isolate 109 segment 11, complete sequence, NCBI accession number is: KF712485.1), and 100% consistency was verified according to the alignment results. HRP-labeled goat anti-rabbit IgG was purchased from Wuhan Tiande Biotechnology Co., Ltd. Block ACE was purchased from Bio-RAD with the catalog number BUF029.

[0023] Example 1. Preparation of grass carp reovirus VP4 protein antigen 1.1. Construction of GCRV VP4 protein recombinant expression vector: According to the gene sequence of GCRV VP4 protein (gene ID: AND67146.1), the full-length fragment was selected as the antigen, the fragment was optimized according to the preference of Escherichia coli codons, and the nucleotide sequence shown in SEQ ID No. 1 was artificially synthesized (synthesized by Wuhan Tiande Biotechnology Co., Ltd.). The nucleotide sequence shown in SEQ ID No. 1 was connected to the pET-28a vector through the NdeI and XhoI restriction sites to obtain the recombinant expression vector pET-28a-VP4.

[0024] 1.2. Transformation of recombinant expression vector: Take 1~2μL of recombinant expression vector and transform it into competent cells BL21(DE3), place it on ice for 30 minutes, and then heat shock it in a 42℃ water bath for 90s; add an appropriate amount of non-antibiotic LB liquid culture medium, and incubate it at 37℃ and 150rpm for 40 minutes. Apply the mixture to LB solid culture medium with corresponding resistance (such as kanamycin), and place it in a 37℃ constant temperature incubator for overnight culture.

[0025] 1.3. Test of inducible expression conditions of recombinant expression vector: Pick a single clone from the culture dish and add it to 3mL of LB medium with corresponding resistance (such as containing kanamycin), and culture it at 37℃ and 200r / min for 3-4h. Take 1.5mL of bacterial solution to a new 15mL centrifuge tube, add 1.5μL IPTG (final concentration is 1mM), and induce culture at 37℃ and 200r / min for 5h. Add 1.5μL IPTG to the remaining 1.5mL bacterial solution, and induce culture at 16℃ and 200r / min overnight. Collect the inducible bacterial solution into 1.5mL centrifuge tubes, centrifuge at 12000g centrifugal force for 1min, and discard the supernatant. Add 1 mL of 1×PBS buffer to the precipitate, and sonicate to lyse the cells; then centrifuge at 12000g for 1 min, take 100 μL of the supernatant and mark it as "NPE", discard the excess supernatant, add 100 μL of 1×PBS (containing 8M urea) to the precipitate, resuspend and mix, and mark it as "DPE". Add 25 μL of 5× reducing loading buffer to the resuspended suspension, boil for 10 minutes, and perform 12% SDS-PAGE detection.

[0026] According to the results of the above expression test, the optimal expression conditions of VP4 protein were determined (see Table 1), and then the expression was amplified.

[0027] Table 1 Optimal expression conditions of VP4 protein

[0028] 1.4. Amplify expression: Take the correctly constructed recombinant expression vector pET28a-VP4 and induce expression according to the optimal expression conditions obtained in step 1.3. Then centrifuge at 4°C and 10,000 rpm for 5 minutes to collect the bacteria.

[0029] 1.5. Purification of VP4 protein: Use an appropriate amount of lysis buffer (PBS with a pH of 7.5, 10% glycerol) to lyse the bacterial cells collected in step 1.4. After thorough mixing, use an ultrasonic disruptor to lyse for 20 minutes, then centrifuge at 12,000 g for 10 minutes, separate the supernatant (the supernatant protein mixture to be purified), and retain the precipitate. Add Ni resin carrying a His protein tag to the supernatant mixture, incubate in a shaker at 4°C for 30 minutes, then collect the resin, temporarily retain the penetration solution (FT), and then purify according to the procedure in Table 2 (the elution time for each step is 5 minutes). The supernatant IN, penetration solution FT, wash buffer W1, wash buffer W2, and eluents E1-E6 after the purification process were sampled for 12% SDS-PAGE detection. The results are as shown below. Figure 1According to the test results, the target protein was collected and dialyzed into 1× PBS buffer (pH=7.4), and the protein concentration was detected by ultra-micro spectrophotometer and 12% SDS-PAGE. The results are shown in Figure 2 shown.

[0030] Table 2 VP4 protein purification procedure

[0031] Figure 1 IN indicates supernatant after disruption, FT indicates penetration, W1 indicates wash buffer 1, W2 indicates wash buffer 2, E1~E6 indicates eluent E1-E6, and M indicates protein marker. Figure 1 As can be seen from the above, the eluents E1-E6 all contain the target protein. Figure 2 The upper M indicates protein marker. Figure 2 It can be seen from the above that the protein has high purity and can be used for subsequent experiments.

[0032] From the test results, it can be seen that most of the VP4 proteins finally obtained exist in the supernatant in a soluble form. The sample information of the VP4 protein is shown in Table 3.

[0033] Table 3 Information of prepared VP4 protein samples

[0034] 2. Preparation of VP4 protein monoclonal antibody 2.1. Mouse immunization: 0.8 mg of the prepared VP4 protein antigen was mixed with an equal amount of adjuvant and emulsified, and then immunized 4-6 week old female BALB / c mice, a total of 4 mice, with an immunization dose of 0.2 mg / mouse / time, and the immunization method was subcutaneous injection in the abdomen. The specific operation steps are shown in Table 4 below. The serum collected before immunization was used as a negative serum control. On the tenth day after each immunization, the tail vein of the immunized mice was blood drawn, and the collected serum was tested for antibody titer by indirect ELISA. When the mouse serum titer reached 10 5 At the above time, the antigen without adjuvant was used for booster immunization, and cell fusion was performed on the 3rd day. The results of mouse serum titer test are shown in Table 5.

[0035] Table 4 Immunization schedule for BALB / c mice

[0036] Table 5 BALB / c mouse serum titer test results

[0037] VP4-1 to VP4-4 in Table 5 represent the corresponding immunized mouse numbers. As can be seen from the table, VP4-4 and VP4-1 have the highest titers, so mice numbered VP4-4 and VP4-1 were selected for the subsequent cell fusion step.

[0038] 2.2 Cell Fusion 2.2.1. One week before cell fusion, resuscitate SP2 / 0 cells and passage them normally so that the number of SP2 / 0 cells in the cell suspension is 1×10 7 At the same time, on the 3rd day after the booster immunization with the antigen without adjuvant, the mice were killed by removing the eyeballs and drawing blood, and the collected serum was used as the positive serum control for the post-fusion test. The mice were soaked in a sterile beaker containing 75% ethanol for 5 minutes for disinfection. After absorbing the liquid on the surface of the mice with absorbent paper, the spleen of the mice was removed with a sterile scalpel, and the spleen was placed on the cell filter. The spleen was gently ground with the needle core of a sterile syringe. During the grinding process, the culture medium (incomplete 1640 culture medium) was continuously added to keep the spleen cells moistened. The filtered cells were collected in a sterile tube with a disposable sterile Pasteur pipette, and the spleen cells were repeatedly and gently blown to form a single-cell suspension. Centrifuged at room temperature and 1500r / min for 10 minutes, the supernatant was discarded, and the precipitate was suspended with incomplete 1640 culture medium so that the number of spleen cells in the suspension was 5×10 7 pcs, spare.

[0039] 2.2.2. Perform cell fusion according to the following steps: thoroughly mix 15 mL of the spleen cell suspension prepared above with a disposable sterile Pasteur pipette, then slowly add it along the wall to 15 mL of pre-prepared SP2 / 0 cells, centrifuge at room temperature at 1500 r / min for 8 minutes; discard the supernatant, gently tap the bottom of the centrifuge tube with your hand to make the precipitate loose and flowable. Equilibrate PEG 1450 (polyethylene glycol 1450) to room temperature in the dark, place the centrifuge tube in a 37°C water bath, and add 1 mL of PEG1450 at a uniform speed within 1 minute, gently shaking the centrifuge tube while adding. Add 1mL of incomplete 1640 medium preheated at 37℃ at a uniform speed within 1min, then add 3mL of incomplete 1640 medium preheated at 37℃ at a uniform speed within 3min, and finally add 10mL of incomplete 1640 medium preheated at 37℃ at a uniform speed within 10min, gently shake the centrifuge tube while adding, and let it stand in a 37℃ water bath for 10min after the addition is completed. Then centrifuge at 800r / min and room temperature for 10min, discard the supernatant, resuspend the cells with 5mL of HAT complete medium containing 10% FBS (fetal bovine serum), and then add HAT complete medium to 50mL. Use a spray gun to add the cell suspension to a 96-well cell culture plate, 100μL / well, then add 100μL / well of HAT complete medium, and place it in a 5% CO 2Culture in a 37℃ cell culture incubator. On the 4th day after fusion, observe the cell status and replace half of the medium, that is, discard 100μL of the medium in the well and add 100μL of fresh HAT complete medium containing 10% FBS. On the 8th to 10th day after fusion, observe the cell status under a microscope, mark the wells containing cell clusters, and perform indirect ELISA to detect whether the cell culture supernatant contains specific antibodies.

[0040] 2.2.3. The operation steps of the indirect ELISA detection method are as follows: (1) Antigen coating: dilute the prepared C-terminal antigen immune complex of VP4 protein with coating solution (50mM CB buffer, pH 9.6) at 10μg / mL, add 100μL to each well, and place in a refrigerator at 4℃ overnight. Discard the liquid in the plate, shake off the water, add 200μL PBST (PBS buffer containing 0.1% (v / v) Tween-20, pH 7.4) to each well, place on a micro-oscillator and shake for 60s, shake off the water, and wash the plate with PBST 3 to 5 times. (2) Blocking: Add blocking solution (1× PBS buffer containing 1wt% BSA and 0.1% (v / v) Tween-20) to the well at 100μL / well and block at 4℃ overnight. Discard the liquid in the plate, spin dry, add PBST at 200 μL / well, spin dry, and wash the plate 3 to 5 times with PBST. (3) Primary antibody incubation: Add 100 μL of the supernatant of the fusion well cells prepared in step 2.2.2, as well as the positive serum control (diluted 32,000 times with blocking solution), the negative serum control (diluted 100 times with blocking solution), and the blank control (no serum, only blocking solution) to the ELISA plate per well and incubate at 37°C for 70 min. Discard the supernatant of the fusion well cells in the plate, spin dry, add PBST at 200 μL / well, spin dry, and wash 3 to 5 times with PBST. (4) Secondary antibody incubation: Dilute HRP-goat anti-mouse IgG with PBS buffer at 1:10,000 (volume ratio), add 100 μL of the diluted secondary antibody to each well, and incubate at 37°C for 60 min. Discard the secondary antibody in the plate, shake off the water, add PBST at 200μL / well, shake off the water, and wash the plate 3 to 5 times with PBST. (5) Color development: Add 100μL 0.32μmol / L TMB (3,3',5,5'-tetramethylbenzidine) color development solution to each well and allow to react at 37°C for 15 minutes (protect from light). (6) Stop the reaction: Add 50μL 2mol / L sulfuric acid to each well to stop the reaction, and read the OD value using an ELISA reader as soon as possible. 450nm Value. OD of the supernatant sample of fusion cells 450nm The OD values ​​of negative serum control samples 450nm A ratio greater than 2.1 was considered positive.

[0041] 2.3 Monoclonal antibody screening and monoclonal hybridoma cell line construction Select the fused positive cell clone wells in the 96-well cell culture plate in step 2.2, wait until all spleen cells die, the fused positive cell clone colonies grow to 1 / 3 to 1 / 2 of the bottom of the well, and the cell culture medium is changed 3 times to make the antibodies secreted by the original spleen cells completely disappear, then aspirate the hybridoma cell culture supernatant 3 to 4 days after the medium is changed or when the cell culture medium turns yellow, and use indirect ELISA to detect positive hybridoma cell colonies. After limiting dilution method subcloning for more than 3 times, when all clone cell colony clone wells are detected as positive, it indicates that this clone is a monoclonal clone and the antibody secreted by this cell line is a monoclonal antibody. Finally, 5 monoclonal cell lines that can stably secrete antibodies were obtained, namely 6E7B3, 6E7B12, 6H5, 6A5B1, and 6A5C4. Among them, 6E7B12 has the highest titer. The OD of ELISA detection after the monoclonal cell line is stable 450nm The values ​​are shown in Table 6.

[0042] Table 6 ELISA detection values ​​of cell culture supernatants of 5 monoclonal cell lines after stabilization

[0043] 2.4 Monoclonal Antibody Purification and Subtype Identification 2.4.1. Preparation of ascites: 8-week-old female BALB / c mice were selected and intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Seven days later, each mouse was intraperitoneally injected with 6 × 10 6 The hybridoma cells prepared in step 2.3 in the logarithmic growth phase were collected from the ascites when the mouse abdomen was observed to be significantly swollen and the fluctuation was obvious when pressed. The ascites was centrifuged at 6000r / min for 10min to remove the liquid paraffin and fat, and the supernatant was aspirated to obtain the mouse ascites antibody.

[0044] 2.4.2. Use Hitrap Protein G HP immunoaffinity chromatography column to purify mouse ascites, the steps are as follows: (a) Dilute the ascites 8-10 times with binding buffer (0.01M PBS solution) and place it on the upper layer of a refrigerator at 4°C for later use. (b) Add 60-80μL Tris-HCl (1mol / L, pH=9.0) to the collection tube to prevent the protein in the ascites from being affected. (c) Rinse the purification column with ultrapure water to remove the ethanol in the column, and control the flow rate at 0.5-1mL / min. (d) Rinse the purification column with 10mL binding buffer to balance the purification column, and control the flow rate at 0.4-0.7mL / min. (e) Use a syringe to add 10mL of the ascites diluent treated in step (a), and control the flow rate at 0.2-0.4mL / min. (f) Rinse the purification column with 10mL binding buffer to wash away unbound impurities, and repeat this step once. (g) Elute with 5 mL of elution buffer (0.1 M Gly-HCl solution, pH 2.5-3.0) at a flow rate of 0.2 mL / min. Collect the eluate in a collection tube to obtain the purified monoclonal antibody.

[0045] 2.4.3 Monoclonal antibody detection and subtype identification (A) The purified monoclonal antibody was detected using 12% SDS-PAGE. Figure 3 As shown. Figure 3 It can be seen from the above that Figure 3 It can be seen from the above that the purity of the purified antibody is above 85%, and the purification effect is good.

[0046] (B) Monoclonal antibody subtype identification: The mouse IgG antibody subtype identification kit (Detai Biotechnology, DTT03) was used for analysis. The purified monoclonal antibody was diluted 8000 times with the diluent provided by the kit and then tested. The operation was carried out strictly according to the instructions of the kit. The results showed that 6E7B12 was an IgG1 antibody and its light chain was a κ chain.

[0047] 3. Preparation, screening and validation of VP4 protein polyclonal antibodies 3.1. Preparation of VP4 protein polyclonal antibodies Take 1.6mg / 1mL of the VP4 protein prepared in step 1.5 as an immunogen to immunize two New Zealand white rabbits. Add an equal amount of complete Freund's adjuvant for emulsification during the first immunization, and add an equal amount of incomplete Freund's adjuvant for the next three times. The emulsified immune complex is subcutaneously immunized at multiple points on the neck and back of 4-month-old male New Zealand white rabbits, and the immunization dose is 800μL / rabbit. The second immunization was performed on the 14th day, the third immunization was performed on the 28th day, and the fourth immunization was performed on the 42nd day. Blood was collected from the neck vein on the 45th day, and the collected blood was stored at 4°C overnight. The blood stored overnight was centrifuged at 5000r / min for 10min, and the serum was collected. The polyclonal antibodies in a portion of the serum were purified by the caprylic acid-ammonium sulfate salting-out method, and the steps are as follows: Take 5mL of serum and centrifuge it at 10000r / min and 4°C for 10min to remove impurities. Take the pretreated serum and add 10mL of 0.06M acetic acid solution with a pH of 5.0, and adjust the pH to 4.8 with 1M HCl; add octanoic acid dropwise at a ratio of 11μL octanoic acid per milliliter of diluted serum under stirring at room temperature, and add it within 30min, and let it stand at 4℃ for 2h. Then centrifuge at 13000r / min for 30min, discard the precipitate, and add 0.01M PBS buffer accounting for 1 / 10 volume of the supernatant after sieving the supernatant through 125μm nylon, and adjust the pH to 7.4 with 1M NaOH. Slowly add an equal volume of saturated ammonium sulfate solution to the serum supernatant while stirring, with a final concentration of 1:1 (V / V), and act at 4℃ for 3h to fully precipitate the protein. Centrifuge at 13000r / min and 4℃ for 10min, discard the supernatant, resuspend the precipitate with 12mL 0.01M PBS buffer, and slowly add 8mL saturated ammonium sulfate solution while stirring, and act at 4℃ for 1h. Centrifuge for 10 min at 13000 r / min and 4°C, discard the supernatant, resuspend the precipitate with 13.3 mL 0.01 M PBS buffer, slowly add 6.7 mL saturated ammonium sulfate solution while stirring, and act at 4°C for 1 h. Centrifuge for 10 min at 13000 r / min and 4°C, discard the supernatant, resuspend the precipitate with an appropriate amount of 0.01 M PBS buffer and place it in a dialysis bag with a permeability of 5 KDa, place the dialysis bag in 50 times the volume of 0.01 M PBS buffer, stir on a magnetic stirrer for 6 h, replace the dialysate 3 times during this period to fully remove the ammonium sulfate. Recover the dialyzed product and centrifuge to obtain the purified polyclonal antibody.

[0048] 3.2 Screening and validation of VP4 protein polyclonal antibodies Use indirect ELISA (refer to the indirect ELISA detection method in step 2.2.3, replace the primary antibody with rabbit antiserum) to detect the titer of the polyclonal antibody in the other part of the serum collected in step 3.1. 450nm Absorbance value / OD of negative serum control 450nmThe absorbance value>2.1 is considered positive. The titer test results of the polyclonal antibody are shown in Table 7.

[0049] Table 7 Results of indirect ELISA testing of polyclonal antibody titers

[0050] From the results in the table, it can be seen that the titer of the obtained serum can reach 1:512000, indicating that a high-titer specific polyclonal antibody was prepared using VP4 protein as an immunogen.

[0051] 3.3. Polyclonal Antibody Western-Blot Verification Add 2 times the volume of RIPA lysis buffer (strong) (Shanghai Biyuntian Biotechnology Co., Ltd., product number P0013B) to the type II GCRV virus isolated from the infected wild grass carp to obtain the virus lysis solution. Take 1μL, 2μL, 4μL, 8μL, and 16μL of the virus lysis solution for detection, respectively. The primary antibody is the antiserum P4-1# and antiserum P4-2# in Table 7, and the dilution concentration is 1:1000 (volume ratio); the secondary antibody is HRP-labeled goat anti-rabbit IgG (H+L), and the dilution concentration is 1:10000 (volume ratio). Chemiluminescence (ECL) color development and photography are performed, and the results are as follows: Figure 4 As shown. Figure 4 It can be seen from the figure that there is a specific band between 60 and 75 kDa, and the size of this band is equivalent to the theoretical molecular weight of VP4 protein, 68.3 kDa.

[0052] 4. Establishment of antibody sandwich ELISA detection method for GCRV VP4 protein 4.1. The steps of antibody sandwich ELISA reaction are as follows: (I) Coat the deep well plate with the purified polyclonal antibody prepared above, 100 μL per well, at 4°C overnight. (II) Add blocking solution, 250 μL per well, and incubate at 37°C for 2 h. (III) Add VP4 protein to the deep wells, 50 μL per well. (IV) Continue to add enzyme-labeled monoclonal antibody and secondary antibody (enzyme-labeled monoclonal antibody is 6E7B3-HRP or 6E7B12-HRP, and secondary antibody is HRP-labeled goat anti-mouse IgG (H+L)) to the deep wells, 100 μL of each antibody per well, and incubate at 37°C for 1 h. (V) Add 0.32 μmol / L TMB color development solution to the deep wells, 100 μL per well, and color develop for 10 minutes. (VI) Then add 2 mol / L sulfuric acid to each well at a volume of 50 μL / well to terminate the reaction, and measure the OD 450nmReaction value. Wash the plate 3 to 5 times with PBST between each step (I) to (V), each time for 90 seconds. Process the data of each test, calculate the P / N value of the reaction, and use the reaction condition with the largest P / N value as the basis for judging the optimal reaction condition of ELISA. The calculation formula of P / N value is as follows: P / N value = OD of positive control well 450nm Mean / negative control well OD 450nm Mean.

[0053] 4.2 Screening of optimal working concentrations of monoclonal and polyclonal antibodies The polyclonal antibody obtained in step 3.2 was diluted from 1:200 to 1:25600 by volume with coating solution (50mM CB buffer, pH 9.6). The two purified and labeled enzyme-labeled monoclonal antibodies with the highest titer in Table 6 were selected and diluted from 1:200 to 1:6400 by volume with blocking solution. According to the method in step 4.1, the chessboard array titration method was used for detection, and the OD450nm value was read with a microplate reader to determine the optimal binding dilution of the two. The results are shown in Table 8.

[0054] Table 8 Results of ELISA array titration of polyclonal and monoclonal antibodies at different concentrations

[0055] From the data in the table, it can be seen that when the polyclonal antibody dilution factor is 1:3200 and the 6E7B3-HRP monoclonal antibody dilution factor is 1:800, the curvature of the change of the P / N value of the reaction is higher than other dilutions, so the polyclonal antibody dilution factor of 1:3200 and the 6E7B3-HRP monoclonal antibody dilution factor of 1:800 are determined to be the best reaction dilutions. When the polyclonal antibody dilution factor is 1:3200 and the 6E7B12-HRP monoclonal antibody dilution factor is 1:1600, the curvature of the change of the P / N value of the reaction is higher than other dilutions, so the polyclonal antibody dilution factor of 1:3200 and the 6E7B12-HRP monoclonal antibody dilution factor of 1:1600 are determined to be the best reaction dilutions.

[0056] 4.3. Screening for the best detection antibody According to the method in step 4.1, ELISA detection was performed using 6E7B3-HRP and 6E7B12-HRP as detection antibodies and antiserum P4-2# as capture antibody. The experimental results are shown in Table 9.

[0057] Table 9 Detection antibody screening results

[0058] It can be seen from the data in the table that the background value of 6E7B12-HRP monoclonal antibody is lower, so 6E7B12-HRP monoclonal antibody is selected as the best detection antibody for making a standard curve.

[0059] 4.4. Screening of blocking solution According to the method in step 4.1, 6E7B12-HRP with a dilution factor of 1:1600 was selected as the detection antibody, and antiserum P4-2# with a dilution factor of 1:3200 was selected as the capture antibody. PBST buffer (1×PBS buffer containing 0.1% (v / v) Tween-20) containing different concentrations of BSA, Block ACE and gelatin was used as the blocking solution for the reaction. ELISA detection was performed. The results are shown in Table 10.

[0060] Table 10 Blocking solution screening results

[0061] The percentages in Table 10 are all mass percentages. From the data in the table, it can be seen that 1wt% BSA + 0.04wt% Block ACE has the best blocking effect. Considering the N value, 1× PBS buffer containing 0.1% (v / v) Tween-20, 0.5wt% BSA and 0.04wt% Block ACE was selected as the blocking solution.

[0062] 4.5. Establishment of standard curve for double antibody sandwich ELISA detection Select 6E7B12-HRP with a dilution factor of 1:1600 as the detection antibody, antiserum P4-2# with a dilution factor of 1:3200 as the capture antibody, and the blocking solution selected in step 4.4 as the blocking solution. Dilute the VP4 protein prepared in step 1.5 to 32ng / mL, 16ng / mL, 8ng / mL, 4ng / mL, 2ng / mL, and 1ng / mL, respectively, and make 2 parallels for each concentration. Perform ELISA detection according to the method in step 4.1, and the standard curve results are shown in Tables 11 and Figure 5 .

[0063] Table 11 Detection results of VP4 protein at different concentrations

[0064] from Figure 5 It can be seen from the figure that when the VP4 protein concentration is 1-32 ng / mL, the VP4 protein concentration and OD 450nm The values ​​have a good linear relationship, and the linear regression equation is y=0.034x+0.0428 (R 2 =0.9912). The horizontal axis x is the VP4 protein concentration in ng / mL; the horizontal axis y is the OD 450nm It can be seen that the detection range of VP4 protein by double antibody sandwich ELISA is 1-32 ng / mL, and the detection limit is 1 ng / mL.

[0065] In summary, the method for double antibody sandwich ELISA detection of VP4 protein using the VP4 protein monoclonal antibody in this application as the detection antibody and the VP4 protein rabbit polyclonal antibody as the capture antibody is as follows: S1. Polyclonal antibody coating: Dilute the VP4 protein polyclonal antibody at a volume ratio of 1:3200 with coating solution (50 mM CB buffer, pH 9.6), then add 100 μL / well into the deep-well plate and place in a 4°C refrigerator overnight.

[0066] S2. Blocking: A mixture of 0.5% BSA and 0.04% Block ACE was used as blocking solution, added to the deep-well plate at a volume of 250 μL / well, and incubated at 37°C for 2 h.

[0067] S3. Add antigen: Add VP4 protein to the deep-well plate at a volume of 50 μL / well. The final concentration of VP4 protein is 1 to 32 ng / mL.

[0068] S4. Add enzyme-labeled secondary antibody: Dilute HRP-labeled 6E7B12 monoclonal antibody (6E7B12-HRP) to 1:1600 (volume ratio) with blocking solution (0.5% BSA + 0.04% Block ACE), add 100 μL / well to the deep-well plate, and incubate at 37°C for 2 hours.

[0069] S5. Substrate color development: Add 0.32 μmol / L TMB color development solution into the deep-well plate at a volume of 100 μL / well and color development for 10 min.

[0070] S6. Termination of reaction: Add 2 mol / L sulfuric acid to the deep-well plate at a volume of 50 μL / well to terminate the reaction, and measure the absorbance value (OD) of the reaction system at 450 nm. 450nm ).

[0071] Wash the plate 3 to 5 times with PBST between each step of steps S1 to S5, each time for 90 s.

[0072] The above-described embodiments are only preferred embodiments of the present application and are not intended to limit the scope of protection of the present application. For any person skilled in the art, the present application may have various changes and modifications. Any simple equivalent changes and modifications made based on the scope of protection of the present application and the contents of the specification should be included in the scope of protection of the present application.

Claims

1. An immunogenic grass carp reovirus VP4 protein, characterized in that: The nucleotide sequence encoding the grass carp reovirus VP4 protein is shown in SEQ ID No.

1.

2. A hybridoma cell line secreting monoclonal antibodies against grass carp reovirus VP4 protein, characterized in that: The hybridoma cell line is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024160; the hybridoma cell line secretes the monoclonal antibody against the VP4 protein of grass carp reovirus described in claim 1.

3. Use of the hybridoma cell line according to claim 2 in the preparation of monoclonal antibodies against the VP4 protein of grass carp reovirus.

4. A monoclonal antibody against grass carp reovirus VP4 protein, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line or its successive cell line according to claim 2.

5. The monoclonal antibody according to claim 4, characterized in that The monoclonal antibody is an IgG1 antibody, and its light chain is of κ subtype.

6. Use of the grass carp reovirus VP4 protein of claim 1 or the hybridoma cell line of claim 2 or the monoclonal antibody of claim 4 or the monoclonal antibody of claim 5 in the preparation of a product for detecting grass carp reovirus.

7. The use according to claim 6, characterized in that: The product for detecting grass carp reovirus is a test kit.

8. An ELISA kit for detecting grass carp reovirus, characterized in that: The ELISA kit comprises the monoclonal antibody described in claim 4 or 5.

9. The ELISA kit according to claim 8, characterized in that The ELISA kit comprises a polyclonal antibody against the VP4 protein of grass carp reovirus as a capture antibody; and the monoclonal antibody described in claim 4 or 5 is used as a detection antibody after being labeled with an enzyme.

10. The ELISA kit according to claim 9, characterized in that The ELISA kit comprises a blocking solution, which is a 1×PBS buffer containing 0.1% (v / v) Tween-20, 0.5wt% BSA and 0.04wt% Block ACE; or / and, the ELISA kit comprises the grass carp reovirus VP4 protein described in claim 1 as an antigen, and the coating concentration of the antigen is 1~32ng / mL.

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