A novel antibody against σc protein of duck reovirus and application thereof

Abstract

The application discloses an antibody for detecting a novel duck reovirus (NDRV), and relates to the field of animal disease prevention and treatment.The antibody comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain is shown as SEQ ID NO.2, and the amino acid sequence of the light chain is shown as SEQ ID NO.4.The novel duck reovirus sigma C protein antibody can be used for directly and rapidly detecting the NDRV sigma C protein in a quick, sensitive and accurate manner when the novel duck reovirus is detected.The antibody can be used for early diagnosis of the NDRV, epidemiological investigation, and antibody screening in vaccine development, and provides an effective tool for preventing and controlling the novel duck reovirus infection.

Description

Technical Field

[0001] The invention relates to a novel duck reovirus σC protein antibody and application thereof, belonging to the technical field of biological detection. Background Art

[0002] Novel duck reovirus (NDRV) is an emerging pathogen characterized by splenomegaly, hemorrhage, and necrosis. Infection can cause depression, loss of appetite, and high mortality in ducklings. It can also lead to decreased egg production in breeder ducks, reduced weight in broiler ducks, and reduced market acceptance rates for broiler ducks. NDRV can infect waterfowl species such as Cherry Valley ducks, Mallards, Muscovy ducks, and geese. NDRV has also been reported to infect chicken embryos, causing significant lesions in the liver, spleen, and bursa of Fabricius. Infected chicks can develop significant lesions in the bursa, liver, and spleen, leading to illness and even death, causing significant economic losses to China's aquaculture industry.

[0003] Among all proteins encoded by the NDRV sequence, the σC protein is crucial for distinguishing NDRV from Muscovy duck reovirus (MDRV), goose reovirus (GRV), and avian reovirus (ARV). Furthermore, the σC protein is highly antigenic and immunogenic, inducing specific immune responses in the host. Therefore, the σC protein is considered an ideal diagnostic marker for novel duck reoviruses and holds significant application value in virus detection, typing, and vaccine development.

[0004] Since the disease has no obvious specific symptoms in the early stages of infection, serological and molecular biological diagnostic methods are commonly used to detect viral antigens or antibodies. The double-antibody sandwich ELISA method uses two specific antibodies to capture the target antigen, which can significantly improve the specificity and sensitivity of the test and is particularly suitable for the detection of low-concentration antigens. However, most existing ELISA methods rely on the whole σC or σB antigens and are highly non-specific. Therefore, there is an urgent need for a detection method that can achieve high sensitivity and specificity for NDRV virus, providing a powerful tool for early detection of NDRV, epidemic control, and vaccine development. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a novel duck reovirus σC protein antibody and its application.

[0006] Technical solution: The present invention provides a novel duck reovirus σC protein antibody, which comprises a heavy chain and a light chain. The amino acid sequence of the heavy chain is shown in SEQ ID NO.2, and the amino acid sequence of the light chain is shown in SEQ ID NO.4.

[0007] Furthermore, the nucleotide sequence encoding the heavy chain is shown as SEQ ID NO.1, and the nucleotide sequence encoding the light chain is shown as SEQ ID NO.3.

[0008] Furthermore, the heavy chain and light chain are connected by a disulfide bond.

[0009] The present invention also provides the use of the novel duck reovirus σC protein antibody in preparing a reagent for detecting the novel duck reovirus.

[0010] The present invention also provides a kit for detecting the novel duck reovirus, wherein the capture antibody in the kit is the novel duck reovirus σC protein antibody.

[0011] Furthermore, the coating concentration of the capture antibody is 100 ng / well.

[0012] Furthermore, the kit also contains novel duck reovirus detection antibodies, novel duck reovirus σC protein standards, negative controls, coating fluid, blocking fluid, diluent, enzyme-labeled secondary antibody, TMB color development fluid, and reaction termination fluid.

[0013] Furthermore, the novel duck reovirus detection antibody is prepared by the following steps: the novel duck reovirus detection antibody is prepared by the following steps: for the first immunization, the novel duck reovirus σC protein is mixed and emulsified with an equal volume of Freund's complete adjuvant; for the second and third immunizations, the novel duck reovirus σC protein is mixed and emulsified with an equal volume of Freund's incomplete adjuvant; each immunization is two weeks apart, and the antibodies are injected into the rabbit through the subcutaneous route on the back, with an injection dose of 1 mg / rabbit; the serum is collected, and purified to obtain rabbit polyclonal antibodies.

[0014] Furthermore, the novel duck reovirus σC protein standard is prepared by the following steps: using the novel duck reovirus full genome as a template, using primers to amplify the σC protein-specific fragment sequence shown in SEQ ID NO.5, constructing a recombinant expression vector, transforming it into BL21 (DE3) competent cells, and inducing with IPTG to obtain the σC protein standard.

[0015] Furthermore, the dilution of the detection antibody is 1:500.

[0016] Furthermore, the blocking solution is 5% skim milk, and the enzyme-labeled secondary antibody is HRP-labeled goat anti-rabbit IgG.

[0017] Beneficial effects: Compared with the prior art, the novel duck reovirus sigma C protein antibody of the application has the following outstanding advantages: the novel duck reovirus sigma C protein antibody of the application can directly target the NDRV sigma C protein for rapid, sensitive and accurate detection when used for detecting the novel duck reovirus. It can be used for early diagnosis of NDRV, antibody screening in epidemiological investigation and vaccine development, and provides an effective tool for preventing and controlling duck novel reovirus infection. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Schematic diagram for identification of SDS-PAGE of GST-sigma C protein expression in the implementation of the application. Lane 1 is a control before induction of the recombinant bacteria, lane 2 is the induced recombinant bacteria, lane 3 is the supernatant of the recombinant bacteria lysate, lane 4 is the precipitate of the recombinant bacteria lysate, lane 5 is a control before induction of the PGEX-6P-1 empty vector, and lane 6 is the induced PGEX-6P-1 empty vector, with an IPTG final concentration of 0.5 mM;

[0019] Figure 2 Schematic diagram for identification of SDS-PAGE of GST-sigma C protein purification in the implementation of the application. Lane 1 is the unpurified GST-sigma C protein, and lane 2 is the purified GST-sigma C protein;

[0020] Figure 3 Western blot characterization of sigma C E10 mouse monoclonal antibody in the implementation of the application;

[0021] Figure 4 Specificity identification of sigma C E10 mouse monoclonal antibody;

[0022] Figure 5 Indirect immunofluorescence identification (IFA) of the purified rabbit polyclonal antibody.

[0023] Figure 6 Specificity detection test of the double antibody sandwich ELISA method. DETAILED DESCRIPTION

[0024] The technical solutions of the application will be further described below in combination with the drawings.

[0025] Sources of materials used in the examples:

[0026] Reagents: SP2 / 0 cell lines and NDRV virus (GenBank accession number: PP934533.1) were maintained in the laboratory. The prokaryotic expression vector pGEX-6P-1 was purchased from Invitrogen. Restriction endonucleases Bam HI and Hind Ⅲ Ⅰ were purchased from New England Biolabs, and protein pre-stained markers were purchased from Thermo Fisher Scientific. Dulbecco's modified Eagle's medium, penicillin-streptomycin, and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco. TMB colorimetric reagent and T4 DNA ligase were purchased from Thermo Fisher Scientific. SDS-PAGE protein loading buffer (5X) was purchased from Invitrogen. HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were purchased from Beyotime.

[0027] Instruments: PCR instrument was purchased from Biori Technology Co., Ltd.; gel imaging system was purchased from Tianneng Technology Co., Ltd.; ultra-clean workbench was purchased from Suzhou Purification Equipment Co., Ltd.; constant temperature metal bath was purchased from Lanbao Experimental Instrument Co., Ltd.; constant temperature incubator and electric constant temperature water bath were purchased from Jinghong Experimental Equipment Co., Ltd.; CO2 incubator was purchased from Thermo Fisher Scientific; high-speed refrigerated centrifuge was purchased from Beckman; Milli-Q low-pyrogen water purifier and electrophoresis instrument were purchased from Millipore.

[0028] Example 1: Construction, protein expression and purification of recombinant plasmid pGEX-6P-σC

[0029] The conserved sequences of NDRVσC gene (GenBank accession number: PP934533.1) were analyzed, and the sequences with high specificity compared with ARV, MDRV and GRV were selected from the conserved sequences. Finally, the sequence shown in SEQ ID NO.5 was selected as the specific fragment (GTTACGGTCTTAGAACGATCGGGTGGTGCGCCGACGCAGTTTGAAGC TCCCTTGCAACTACAAAACGGAGTCGTCTCACTCCAAGCATCTCCCTCTTTCTGTTCTTTGTCTCCGATCCTCTCCGGACCTGCTGATGCTGCTGTCTTCAAGGTTGGTGAGTGGCTGGGAACTGTTATATCTGGTCAAAGTCAGTCATCTGCAATTATGAACGTGCGGATTCATTCATTTGGGCAGCGGACCATGTTGCTTATGTCTTCGCAAAATGTATTCACTATTCCGCCAGGTTCGGGTGCGTCTTTGCAGCTAGATGTGACTCGCATAACGACCCCTGCCATTGACGTTGCTATGGTAACTCCTTCTGCT). A pair of primers were designed for this sequence using Primer Primer 5 software. The primers were synthesized by Qingke Biotechnology Co., Ltd. The primer sequences are shown in SEQ ID NO. 6-7: F: 5'-ATA GGATCC GTTACGGTCTTAGAACGATCGGG-3' (underlined: Bam HI restriction site), R: 5'-ATT CTCGAG AGCAGAAGGAGTTACCATAGCAA-3' (the underline is the Hind Ⅰ Ⅰ Ⅰ restriction enzyme site). The amplified target fragment size is 381bp. Next, the RNA of the novel duck reovirus was extracted and reverse transcribed into cDNA using the extracted RNA as a template. The reverse transcription system was: Random Primer (6nt) 2uL, 2.5mM dNTPs 4uL, RT 1µL, Ribonuclease Inhibitor 0.5µL, 5× ES RT Buffer 4µL, RNA template 8.5µL. Reverse transcription was performed at 25°C for 5min, 42°C for 30min, and 85°C for 5s. The resulting cDNA was amplified using the aforementioned primers using PCR. The reaction system consisted of ddH₂O 13.4µL, 10× Taq Buffer 2µL, EasyTaq DNA polymerase 0.2µL, Forward Primer 0.4µL, Reverse Primer 0.4µL, and 2.5mM dNTPs 1.6µL. The PCR amplification program was as follows: a pre-denaturation phase at 95°C for 5min; followed by a cyclic amplification phase consisting of 35 cycles of denaturation at 95°C for 30s, annealing at 56°C for 30s, and extension at 72°C for 30s; and finally, extension at 72°C for 10min. Electrophoresis analysis was performed on agarose gel with a concentration of 1%. The samples identified as positive by agarose gel electrophoresis were recovered from the gel, and the PCR products and pGEX-6P-1 vector purified by gel recovery were double-digested with HindⅠⅠⅠ and BamHⅠ, respectively. Subsequently, the target fragment was cloned into the prokaryotic expression vector pGEX-6P-1 using T4 DNA ligase to obtain the recombinant cloning vector pGEX-6P-σC. The recombinant plasmid pGEX-6P-σC was digested and identified using HindⅠⅠⅠ and Bam HⅠ, and a 4984bp band and a 381bp band were obtained. The recombinant plasmids were confirmed by DNA sequencing and then transferred into the host bacteria BL21 (DE3). After induction with IPTG at 37℃ for 5h, the mixture was centrifuged and ultrasonically broken to separate the supernatant and precipitate. The expression of the recombinant protein was analyzed by SDS-PAGE (such as Figure 1 The results showed that the expressed σC recombinant protein was mainly expressed in inclusion bodies, with a protein size of approximately 38.5 kDa, which was consistent with the expected size. Finally, the protein was purified and analyzed by SDS-PAGE. The protein concentration was determined using a BCA kit. The results showed that the purified GST-σC protein had a single band and high purity (such as Figure 2 ).

[0030] Example 2: Preparation and identification of NDRV-σC protein-specific monoclonal antibodies

[0031] (1) Immunization of BALB / c mice with σC protein

[0032] Four-week-old female BALB / c mice (provided by the Center for Comparative Medicine, Yangzhou University) were inoculated subcutaneously on their backs with 100 μg of purified GST-σC protein emulsified with an equal amount of recombinant antigen in complete Freund's adjuvant. Booster immunizations were performed two times every two weeks; the booster immunization used recombinant antigen emulsified with purified GST-σC and an equal amount of incomplete Freund's adjuvant. Two weeks after the second booster immunization, blood was collected to measure serum antibody levels. Mice with antibody titers exceeding 100,000 were given a booster immunization with 100 μg of purified GST-σC antigen intraperitoneally. Three days later, antibody-secreting splenic lymphocytes were prepared.

[0033] (2) Preparation and identification of σC monoclonal antibodies

[0034] (2.1) Cell fusion

[0035] ① Select the mouse with the highest antibody titer, kill it by cervical dislocation, and disinfect it in 75% alcohol for 5 minutes. Then transfer it to a biosafety cabinet. Fix the mouse's limbs with foam boards, remove the spleen, place it on a copper grid, and slowly add DMEM medium while gently pressing to make a spleen cell suspension. Collect it into a 50mL centrifuge tube.

[0036] ② Take myeloma cells (SP2 / 0 cell line) grown to the logarithmic growth phase and mix them with spleen cells. Then, slowly add 1 mL of PEG solution dropwise within 1 minute in a 40°C water bath, gently stirring while adding, and then let it stand for 30 seconds. After the standing period, add 1 mL of DMEM medium dropwise within 1 minute while stirring. Add 30 mL of DMEM medium within 6 minutes to terminate the fusion.

[0037] ③ Centrifuge at 1000 rpm for 10 min, discard the supernatant, add 30 mL of HAT medium to the centrifuge tube, resuspend the cell pellet, add the cell suspension in a volume of 100 μL to the prepared feeder cells (mouse peritoneal macrophages), and culture in a 37°C CO2 incubator.

[0038] ④ On the 4th and 8th days of static culture, replace 2 / 1 of the medium in the well with HAT medium containing 20% ​​FBS. On the 10th day, replace all the original HAT medium in the well with HT medium containing 20% ​​FBS. When the medium in the well begins to turn yellow, it can be aspirated for testing.

[0039] (2.2) Screening and subcloning of positive hybridoma cells

[0040] Screening of positive hybridoma cells: Immunized mouse serum was used as the positive control and non-immunized mouse serum as the negative control. The cells were detected by indirect ELISA. When the OD value of the cell supernatant was greater than 2.1 times that of the negative control, the cells were considered positive.

[0041] Hybridoma cell subcloning: Positive hybridoma cell lines with high OD values ​​were selected and plated onto 96-well plates by limiting dilution, ensuring that only a single hybridoma cell was present in each well. After culturing for 7 days in a 37°C, 5% CO2 incubator, the cell supernatant was aspirated and tested for reactivity against the σC protein using an ELISA assay. Wells with high OD values ​​were selected for the next round of subcloning. After three consecutive subcloning cycles, the monoclonal cell line NDRV-σC-E10 was obtained and expanded.

[0042] (2.3) Ascites Preparation

[0043] Multiparous BALB / c female mice were sensitized with 0.5 mL of incomplete Freund's adjuvant intraperitoneally. One week later, 50,000 hybridoma cells were injected intraperitoneally into the female mice. One week later, ascites was collected and its reactivity with the σC protein was determined using an ELISA. The results showed that the σC monoclonal antibody prepared using monoclonal antibody technology showed strong reactivity with the σC protein and could be used to establish a double-antibody sandwich ELISA assay.

[0044] (2.4) Reactivity of monoclonal antibodies with NDRV

[0045] In order to evaluate the reaction characteristics between the monoclonal antibody and NDRV virus, western blot and indirect immunofluorescence (IFA) identification methods were used. The results showed that this monoclonal antibody had WB characteristics (such as Figure 3 ), but without the IFA feature.

[0046] (2.5) Determination of affinity between monoclonal antibodies and σC protein

[0047] Mouse ascites was diluted with PBST at 10 dilutions: 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000. The affinity of the ascites to σC protein was tested by indirect ELISA. The results showed that the monoclonal antibody had a high affinity for σC protein.

[0048] Table 1 Affinity determination of monoclonal antibodies and σC protein

[0049]

[0050] (2.6) Monoclonal antibody specificity determination

[0051] Protein samples of ARV (JN559375.1), MDRV (OK626889.1), NDRV (PP934533.1), and GRV (AJ717738.1) viruses were collected and the reactivity of the monoclonal antibody with the above viruses was determined by western blot. The results showed that the monoclonal antibody only reacted with NDRV, but not with ARV, MDRV, or GRV (e.g. Figure 4 ).

[0052] (2.7) Monoclonal antibody sequencing

[0053] The monoclonal antibody was sequenced, wherein the nucleotide sequence of the heavy chain is shown in SEQ ID NO. 1 (CCTCCTGTCAGTAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCGGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAGGGCTACTGGCTACACATTCAGAAGTTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGAGGCACTACTCACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGTCTACATGCAACTCAGTAGCCTGACATCTGAGGACTCTGCCGTCTTTTACTGTGCAAGGGGGACCCCCTGGTACTACTTTGACTACTGGGGCCAAGGCACCGCTCTCACGGTCTCCTCAGCCAAAACGACACCCCCATC (405 bp)); Shown in NO.2 (FLLSVTAGVHSQVQLQRSGAELMKPGASVKISCRATGYTFRSYWIEWVKQRPGHGLEWIGEILPGRGTTHYNEKFKGKATFTADTSSNTVYMQLSSLTSEDSAVFYCARGTPWYYFDYWGQGTALTVSSAKTTPPS).

[0054] The nucleotide sequence of the light chain is shown in SEQ ID NO.3 (GGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACATTGTGCTGACACAGT CTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCA GGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTG (386bp)); the base acid sequence is SEQ Shown as IDNO.4 (WVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKP GQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGG PSWK).

[0055] The light and heavy chains are linked by disulfide bonds.

[0056] Example 3: Preparation and purification of rabbit polyclonal antibodies against σC protein

[0057] Preparation of rabbit polyclonal antibodies: During the initial immunization, the purified recombinant GST-σC protein prepared in Example 1 was mixed and emulsified with an equal volume of Freund's complete adjuvant, and injected into a healthy rabbit weighing about 2 kg through the subcutaneous route on the back. The injection dose was 1 mg / rabbit. Two weeks after the initial immunization, the purified recombinant GST-σC protein was mixed and emulsified with an equal volume of Freund's incomplete adjuvant. The third immunization was performed two weeks after the second immunization, and the second immunization method was the same. One week after the third immunization, positive serum was collected by cardiac blood sampling, and the antibodies were purified using the caprylic acid-ammonium sulfate precipitation method. The concentration of the purified antibodies obtained was determined by the BCA method, and the antibodies were verified by the indirect immunofluorescence (IFA) method. The results showed that rabbit polyclonal antibodies against NDRVσC protein (such as Figure 5 ).

[0058] Implementation 4: Establishment of double antibody sandwich ELISA method

[0059] 1. Establishment of the Double Antibody Sandwich ELISA Method

[0060] A double-antibody sandwich ELISA method was established using the σC E10 monoclonal antibody prepared in Example 2 as the capture antibody, the purified rabbit polyclonal antibody prepared in Example 3 as the detection antibody, and the GST-σC protein purified in Example 1 as the standard.

[0061] ① Determine the optimal reaction concentrations of capture antibody and detection antibody by checkerboard titration. The purified σC E10 monoclonal antibody was coated at 100, 200, 300, and 400 ng / well, and the ELISA plate was incubated at 4°C overnight. After washing and blocking, 400 ng of purified GST-σC protein was added to each well. PBS was added as a negative control and incubated at 37°C for 1 hour. After washing three times, rabbit polyclonal antibody diluted at 1:500, 1:1000, 1:2000, and 1:4000 was added, 100 μL per well, incubated at 37°C for 1 hour, washed three times, and goat anti-rabbit IgG-HRP enzyme-labeled secondary antibody diluted at 1:5000 was added, 100 μL per well, incubated at 37°C for 1 hour, washed three times with PBST, and 100 μL of TMB color development solution was added to each well. The plate was incubated at room temperature in the dark for 30 minutes, and 1M HCl was added to terminate the color development reaction. The OD was measured using a microplate reader. 450 Calculate the positive and negative OD values 450 The ratio (P / N) of the two groups was the best, and the best dilution combination was determined. The results are shown in Table 2.

[0062] Table 2 Optimal concentrations of capture antibody and detection antibody

[0063]

[0064] The results showed that the P / N value was the largest when the concentration of the capture antibody was 100 ng / well and the dilution ratio of the detection antibody was 1:500. At this time, the concentrations of the capture antibody and the detection antibody were optimal.

[0065] ② Optimization of blocking conditions: 5% skim milk and 3% skim milk were selected as blocking solutions, with 3 replicates per group. After determining the blocking solution concentration, the optimal blocking time was explored. Block at 37°C for 1 h, 2 h, and 3 h, with 3 replicates per group. The OD values ​​were measured. 450 value, calculate P / N to determine the optimal closing time.

[0066] Table 3 Optimal blocking solution concentration and blocking time

[0067]

[0068] The results showed that the P / N value was the largest when 5% skim milk was used as the blocking solution and incubated at 37°C for 3 hours. Therefore, the optimal concentration of the blocking solution was 5% skim milk, and the optimal blocking time was 3 hours.

[0069] ③ Optimization of antigen incubation time: set four antigen incubation times of 30min, 45min, 60min, and 90min respectively, and measure OD 450 The value was calculated and the P / N ratio was used to determine the antigen incubation time.

[0070] Table 4 Optimal incubation time for antigens

[0071]

[0072] The results showed that the P / N value was the largest when the antigen was incubated for 45 minutes, so the optimal antigen incubation time was 45 minutes.

[0073] ④Optimization of enzyme-labeled secondary antibody dilution concentration: dilute goat anti-rabbit IgG-HRP enzyme-labeled secondary antibody at a ratio of 1:5000 and 1:10000, and measure OD 450 The P / N value was calculated to determine the incubation time of enzyme-labeled secondary antibody.

[0074] Table 5 Optimal dilution of enzyme-labeled secondary antibodies

[0075]

[0076] The results showed that when the concentration of enzyme-labeled secondary antibody was 1:10000, the P / N value was the largest, so 1:10000 was the optimal dilution factor of enzyme-labeled secondary antibody.

[0077] 2. Establishment of the double antibody sandwich ELISA method

[0078] ① Coating: Dilute σC E10 mouse monoclonal antibody in carbonate buffer, 100 ng / well, and incubate overnight at 4°C;

[0079] ② Blocking: discard the coating solution, wash each well with PBST three times, pat dry for the last time, add 200 μL 5% skim milk to each well, and block at 37°C for 3 h;

[0080] ③ Antigen incubation: discard the blocking solution, wash each well with PBST three times, pat dry for the last time, add the antigen to be tested, 100 μL / well, and incubate at 37°C for 45 min;

[0081] ④ Detection antibody incubation: discard the antigen, wash each well with PBST three times, pat dry for the last time, add 1:500 diluted rabbit polyclonal antibody, 100 μL / well, and incubate at 37°C for 1 hour;

[0082] ⑤ Secondary antibody incubation: discard the detection antibody, wash each well with PBST three times, pat dry for the last time, add 1:10000 diluted enzyme-labeled secondary antibody (HRP-labeled goat anti-rabbit IgG), 100 μL / well, and incubate at 37°C for 1 hour;

[0083] ⑥ Color development: discard the enzyme-labeled secondary antibody, wash each well with PBST 5 times, add 100 μL TMB color development solution to each well and incubate at 37°C for 25 minutes;

[0084] ⑦ Stop: Add stop solution 1M HCl, 100 μL / well;

[0085] ⑧Reading: Read OD with a microplate reader 450nm value.

[0086] 3. Determination of the critical value of the double antibody sandwich ELISA kit

[0087] 20 SPF duck spleen grinding fluids identified as negative by RT-PCR were selected as samples, and the wells with GST-σC protein added were used as positive controls, and the wells with PBST added were used as negative controls for ELISA detection. The ELISA detection was performed according to the experimental conditions determined above to determine the critical value. The ELISA detection results were judged by calculating the S / P value. The formula is S / P = (sample OD value - average OD value of negative control) / (positive control OD value - average OD value of negative control). The critical value is calculated as: the sum of the arithmetic mean of S / P and 3 times the standard deviation SD. According to the above experimental results, the critical value of the new duck reovirus double antibody sandwich ELISA detection method was determined to be 0.2, and the sample OD 450nm A value greater than 0.2 was considered positive, and OD 450nm A value less than or equal to 0.2 was considered negative.

[0088] 4. Specificity of Double Antibody Sandwich ELISA Kit

[0089] The established double antibody sandwich ELISA method was used to detect positive sera of six pathogens, including ARV (JN559375.1), NDRV (PP934533.1), MDRV (OK626889.1), GRV (AJ717738.1), Tembusu virus (DTMUV) (LN849063.1), and duck hepatitis virus (DHV) (KJ524552.1), and the specificity of the double antibody sandwich ELISA kit was analyzed. The results showed that ( Figure 6 ), the double-antibody sandwich ELISA method established can only detect NDRV and does not react with other common pathogens.

[0090] 5. Sensitivity of the Double Antibody Sandwich ELISA Kit

[0091] The NDRV virus supernatant (TCID 50 =5.62×10 -5 / 0.1mL) was serially diluted at ratios of 1:10, 1:100, 1:1000, 1:10000, and 1:100000, and normal Vero cell culture supernatant was used as a negative control. ELISA detection was performed using the determined conditions. The results showed that the double antibody sandwich ELISA method established in this study could detect a minimum of 5.62×10 -2 TCID 50 .

[0092] Table 6 Sensitivity test

[0093]

[0094] Practice 5: Application of double antibody sandwich ELISA kit for detecting novel duck reovirus

[0095] The double antibody sandwich ELISA method established by the present invention was used to detect 20 liver and spleen tissues collected from a duck farm in Anhui Province that were positive for NDRV virus by RT-PCR. The test results showed that the OD values ​​of the 20 samples detected by this method were 450nm The values ​​were all greater than 0.2, which was 100% consistent with the RT-PCR test results.

[0096] Table 7 Results of clinical sample detection using the novel duck reovirus double antibody sandwich ELISA kit

[0097]

[0098]

Claims

1. A novel duck reovirus σC protein antibody, characterized in that: The antibody comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO.2, and the amino acid sequence of the light chain is shown in SEQ ID NO.

4.

2. The novel duck reovirus σC protein antibody according to claim 1, characterized in that The heavy and light chains are linked by disulfide bonds.

3. The gene encoding the novel duck reovirus σC protein antibody according to claim 1, characterized in that: The nucleotide sequence encoding the heavy chain is shown in SEQ ID NO.1, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO.

3.

4. Use of the novel duck reovirus σC protein antibody according to any one of claims 1 to 3 in the preparation of a reagent for detecting the novel duck reovirus.

5. A kit for detecting a novel duck reovirus, characterized in that: The capture antibody in the kit is the novel duck reovirus σC protein antibody according to any one of claims 1 to 3.

6. The kit for detecting novel duck reovirus according to claim 5, characterized in that: The coating concentration of the capture antibody was 100 ng / well.

7. The kit for detecting novel duck reovirus according to claim 5, characterized in that: The kit also contains novel duck reovirus detection antibody, novel duck reovirus σC protein standard, negative control, coating solution, blocking solution, diluent, enzyme-labeled secondary antibody, TMB color development solution, and reaction termination solution.

8. The kit for detecting novel duck reovirus according to claim 7, characterized in that: The novel duck reovirus detection antibody is prepared by the following steps: for the first immunization, the novel duck reovirus σC protein is mixed and emulsified with an equal volume of Freund's complete adjuvant; for the second and third immunizations, the novel duck reovirus σC protein is mixed and emulsified with an equal volume of Freund's incomplete adjuvant; each immunization is separated by two weeks and injected into rabbits through the subcutaneous route at the back, with an injection dose of 1 mg per rabbit; serum is collected, and purified to obtain rabbit polyclonal antibodies.

9. The kit for detecting novel duck reovirus according to claim 7, characterized in that: The novel duck reovirus σC protein standard is prepared by the following steps: using the novel duck reovirus full genome as a template, using primers to amplify the σC protein-specific fragment sequence shown in SEQ ID NO.5, constructing a recombinant expression vector, and expressing the novel duck reovirus σC protein standard using a prokaryotic expression system.

10. The kit for detecting novel duck reovirus according to claim 7, characterized in that: The dilution of the detection antibody was 1:500.

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