Novel duck reovirus sigma C protein antibody and application thereof

Abstract

The invention discloses an antibody, the antibody comprises a heavy chain and a light chain, 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. When the novel duck reovirus sigmaC protein antibody is used for detecting the novel duck reovirus, the novel duck reovirus sigmaC protein can be directly, rapidly, sensitively and accurately detected in allusion to the NDRV sigmaC protein. The kit can be used for early diagnosis of NDRV, epidemiological investigation and antibody screening in vaccine research and development, and provides an effective tool for preventing and controlling duck novel 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. The main characteristics of the virus include splenomegaly, hemorrhage and necrosis. After infection, ducklings become depressed, have anorexia and have a high mortality rate; they also lead to a decrease in the egg production rate of breeder ducks, a decrease in the weight of broiler ducks and a decrease in the qualified rate of broiler ducks. It can infect waterfowl species such as Cherry Valley ducks, Mallards, Muscovy ducks and geese. In addition, it has been reported that NDRV infects chicken embryos, causing obvious lesions in the liver, spleen, bursa of Fabricius, etc. Infecting chicks can cause obvious lesions in the bursa of Fabricius, liver and spleen, leading to illness and even death, causing huge economic losses to China's breeding industry.

[0003] Among all proteins encoded by the NDRV sequence, the σC protein is an important gene for distinguishing NDRV from Muscovy duck reovirus (MDRV), goose reovirus (GRV) and avian reovirus (ARV). In addition, the σC protein has high antigenicity and immunogenicity, and can induce specific immune responses in the host. Therefore, the σC protein is considered to be an ideal diagnostic marker for the new duck reovirus, and has important application value in virus detection, typing analysis and vaccine development.

[0004] Since the disease has no obvious specific symptoms in the early infection stage, serological diagnostic methods and molecular biological diagnostic methods are often 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 detection, and is particularly suitable for the detection of low-concentration antigens. However, most of the existing ELISA methods rely on σC or σB whole antigens, which are highly non-specific. Therefore, there is an urgent need for a detection method that can achieve high sensitivity and specificity for NDRV virus, which provides 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 in SEQ ID NO.1, and the nucleotide sequence encoding the light chain is shown in SEQ ID NO.3.

[0008] Furthermore, the heavy chain and the light chain are connected via 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 a 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 antibody, novel duck reovirus σC protein standard, negative control, coating solution, blocking solution, diluent, enzyme-labeled secondary antibody, TMB colorimetric solution, and reaction termination solution.

[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 injected into the rabbit subcutaneously on the back with an interval of two weeks; the injection dose is 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 present invention has the following outstanding and significant advantages: the novel duck reovirus σC protein antibody of the present invention can directly detect the NDRV σC protein quickly, sensitively and accurately when used to detect the novel duck reovirus. It can be used for early diagnosis of NDRV, epidemiological surveys and antibody screening in vaccine development, providing an effective tool for preventing and controlling novel duck reovirus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of SDS-PAGE identification of GST-σC protein expression in the implementation of the invention. Lane 1 is the control before induction of 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 the control before induction of PGEX-6P-1 empty vector, lane 6 is the induced bacteria of PGEX-6P-1 empty vector, and the final concentration of IPTG is 0.5mM;

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

[0020] Figure 3 To verify the western blot characteristics of σCE10 mouse monoclonal antibody in the implementation of the present invention;

[0021] Figure 4 For specific identification of σC E10 mouse monoclonal antibody;

[0022] Figure 5 Indirect immunofluorescence analysis (IFA) of purified rabbit polyclonal antibodies.

[0023] Figure 6 It is a specific detection test using double antibody sandwich ELISA method. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0025] Sources of materials used in the examples:

[0026] Reagents: SP2 / 0 cell line and NDRV virus (GenBank accession number: PP934533.1) were stored in the laboratory. Prokaryotic expression vector pGEX-6P-1 was purchased from Invitrogen. Restriction endonucleases Bam HI and Hind ⅠⅠⅠ were purchased from NEB, protein pre-stained markers were purchased from Thermo Fisher Scientific; DMEM medium, penicillin-streptomycin and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco; TMB colorimetric solution and T4 DNA ligase were purchased from Thermo Fisher Scientific; SDS-PAGE protein loading buffer (5X) was purchased from Invitrogen. HRP-labeled goat anti-rabbit IgG and HRP-labeled 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.; clean bench was purchased from Suzhou Purification Equipment Co., Ltd.; constant temperature metal bath was purchased from Blue Leopard Experimental Instrument Co., Ltd.; constant temperature incubator and electric constant temperature water tank 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 conservative sequence of NDRVσC gene (GenBank accession number: PP934533.1) was analyzed, and the sequence with higher specificity compared with ARV, MDRV and GRV was selected from the conservative sequence. Finally, the sequence shown in SEQ ID NO.5 was selected as the specific fragment (GTTACGGTCTTAGAACGATCGGGTGGTGCGCCGACGCAGTTTGAAGC TCCCTTGCAACTACAAAACGGAGTCGTCTCACTCCAAGCATCTCCCTCTTTCTGTTCTTTGTCTCCGATCCTCTCCGGACCTGCTGATGCTGCTGTCTTCAAGGTTGGTGAGTGGCTGGGAACTGTTATATCTGGTCAAAGTCAGTCATCTGCAATTATGAACGTGCGGATTCATTCATTTGGGCAGCGGACCATGTTGCTTATGTCTTCGCAAAATGTATTCACTATTCCGCCAGGTTCGGGTGCGTCTTTGCAGCTAGATGTGACTCGCATAACGACCCCTGCCATTGACGTTGCTATGGTAACTCCTTCTGCT). A pair of primers was designed for this sequence using Primer Primer5 software, and 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 HindⅠⅠⅠ restriction site). The amplified target fragment size is 381bp. Next, the RNA of the new duck reovirus was extracted and reverse transcribed into cDNA using the extracted RNA as a template. The reverse transcription system: Random Primer (6nt) 2uL, 2.5mM dNTPs 4uL, RT 1uL, Ribonuclease Inhibitor 0.5uL, 5×ES RT Buffer 4uL, RNA template 8.5uL. The reverse transcription program was 25℃5min, 42℃30min, 85℃5s. The obtained cDNA was amplified by PCR using the above primers, the reaction system: ddH2O13.4uL, 10×Taq Buffer 2uL, EasyTaqDNA polymerase 0.2uL, Forward Primer 0.4uL, Reverse Primer 0.4uL, 2.5mM dNTPs 1.6uL, PCR amplification program was set as follows: first, the pre-denaturation stage, the temperature was set to 95℃ for 5 minutes; then the cycle amplification stage, each cycle included: 95℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 30 seconds, a total of 35 cycles; finally, extension at 72℃ for 10 minutes. Electrophoresis analysis was performed using agarose gel with a concentration of 1%. 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 4984 bp band and a 381 bp 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°C for 5 hours, the mixture was centrifuged and ultrasonically disrupted 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, and the protein size was about 38.5 kDa, which was consistent with the expected size. Finally, the protein was purified, and the purification effect was analyzed by SDS-PAGE. The protein concentration was determined by 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 of Yangzhou University) were subcutaneously inoculated with 100 μg of purified GST-σC protein and recombinant antigen emulsified with an equal amount of complete Freund's adjuvant on their backs. Booster immunizations were performed every 2 weeks for a total of two booster immunizations; the strong immunizations were performed with recombinant antigens containing purified GST-σC and an equal amount of incomplete Freund's adjuvant emulsified. Two weeks after the second booster immunization, blood was collected to determine the serum antibody level of mice, and mice with an antibody titer of more than 100,000 were intraperitoneally inoculated with 100 μg of purified GST-σC antigen for booster immunization, and three days later, they were used to prepare spleen lymphocytes that secrete antibodies.

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

[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 mesh, press gently while slowly adding DMEM culture medium to make a spleen cell suspension, and collect it in 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 evenly, then slowly add 1 mL of PEG solution in 1 min in a 40°C water bath, gently stir while adding, and then let it stand for 30 seconds. After standing, add 1 mL of DMEM culture medium within 1 min, stir while adding, and add 30 mL of DMEM culture medium within 6 min 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 culture medium in the well with HAT culture medium containing 20% ​​FBS. On the 10th day, replace all the original HAT culture medium in the well with HT culture medium containing 20% ​​FBS. When the culture medium in the well begins to turn yellow, it can be aspirated for detection.

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

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

[0041] Subcloning of hybridoma cells: Select the positive hybridoma cell lines with higher OD values, and plate them on 96-well plates by limiting dilution, so that there is only a single hybridoma cell in each well. After culturing in a CO2 incubator at 37°C and 5% CO2 for 7 days, take the cell supernatant and use ELISA to detect its reactivity against σC protein, and take the wells with higher OD values ​​for the next round of subcloning; after three consecutive subclones, the monoclonal cell line NDRV-σC-E10 was obtained and expanded.

[0042] (2.3) Ascites preparation

[0043] Multiparous BALB / c female mice were selected, and each female mouse was sensitized by intraperitoneal injection of 0.5 mL of Freund's incomplete adjuvant. One week later, 50,000 hybridoma cells were injected into the female mouse via the peritoneal route. One week later, ascites was collected and its reactivity with σC protein was determined by ELISA. The results showed that the σC monoclonal antibody prepared using monoclonal antibody technology had a strong reactivity with σC protein and could be used for the subsequent establishment of a double antibody sandwich ELISA method.

[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, and the results showed that this monoclonal antibody had WB characteristics (such as Figure 3 ), but without the IFA feature.

[0046] (2.5) Determination of the affinity of monoclonal antibodies to σC protein

[0047] The mouse ascites was diluted with PBST at 10 dilutions of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000, and the affinity between the mouse ascites and σC protein was detected 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 between the monoclonal antibody and the above viruses was determined by western blot. The results showed that this monoclonal antibody only reacted with NDRV, but not with ARV, MDRV, and GRV (such as 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 in 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 subcutaneously into a healthy rabbit weighing about 2 kg at 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 immunization method was the same as the second one. 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 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 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 concentration of capture antibody and detection antibody by chessboard titration method. The purified σCE10 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, and PBS was added as a negative control. Incubate at 37°C for 1 hour, wash 3 times, add rabbit polyclonal antibody diluted at 1:500, 1:1000, :2000, and 1:4000, 100 μL per well, incubate at 37°C for 1 hour, wash 3 times, add goat anti-rabbit IgG-HRP enzyme-labeled secondary antibody diluted at 1:5000, 100 μL per well, incubate at 37°C for 1 hour, wash 3 times with PBST, add 100 μL TMB colorimetric solution to each well, incubate the ELISA plate at room temperature and avoid light for 30 minutes, then add 1M HCl to terminate the colorimetric reaction, and use an ELISA reader to measure OD 450 Calculate positive and negative OD values 450 The ratio (P / N) of the two dilutions was the largest, 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, and the concentrations of the capture antibody and the detection antibody were optimal at this time.

[0065] ② Optimization of blocking conditions: 5% skim milk and 3% skim milk were selected as blocking solutions, with 3 replicates in each group. After determining the concentration of the blocking solution, the optimal blocking time was explored. The cells were blocked at 37°C for 1h, 2h, and 3h, with 3 replicates in each group. The OD 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 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 value, calculate P / N and 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] ④ Optimization 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 antibody

[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 multiple of enzyme-labeled secondary antibody.

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

[0078] ① Coating: Dilute σCE10 mouse monoclonal antibody in carbonate buffer, 100 ng / well, 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℃ for 3h;

[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 min;

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

[0085] ⑧Reading: Read OD with an ELISA 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 test 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 S / P arithmetic mean 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 this double antibody sandwich ELISA kit was analyzed. The results showed that ( Figure 6 ), the established double antibody sandwich ELISA method can only detect NDRV and does not react with other common pathogens.

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

[0091] The NDRV virus supernatant (TCID 50 =5.62×10 -5 / 0.1mL) was diluted in a gradient ratio 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 under 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] Implementation 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 new 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 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.

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

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 comprises a novel duck reovirus detection antibody, a novel duck reovirus σC protein standard, a negative control, a coating solution, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a TMB color developing solution, and a reaction stopping 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 the rabbit through the subcutaneous route on the back, the injection dose is 1 mg / rabbit, serum is collected, and the rabbit polyclonal antibody is purified.

9. The kit for detecting a 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 using a prokaryotic expression system to express the novel duck reovirus σC protein standard.

10. The method according to claim 7, characterized in that The dilution of the detection antibody was 1:500.

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