Novel duck reovirus sigma C protein monoclonal antibody

Abstract

The invention belongs to the technical field of biological immunity, and provides a novel duck reovirus sigma C protein monoclonal antibody, the heavy chain of the monoclonal antibody is IgG1 type, and the light chain is Kappa chain. The number of positive hybridoma cells of the monoclonal antibody is three, and the positive hybridoma cells are named as 5B7, 3C10 and 6G11. And the positive hybridoma cell of the monoclonal antibody is 6G11. And the minimum epitope of the monoclonal antibody is 304 to 314 aa. According to the present invention, the NDRV recombinant sigma C protein is expressed, the hybridoma technology is adopted to obtain the monoclonal antibody with high titer and good activity, the monoclonal antibody is adopted to screen and identify the novel antigen epitope 304WQDLVPVWLQ314 of the NDRV, and the epitope has high conservative property in the NDRV strain, and is the specific antigen epitope of the NDRV;

Description

Technical Field

[0001] This invention belongs to the field of bioimmunotechnology, specifically relating to a novel monoclonal antibody against duck reovirus σC protein. Background Technology

[0002] Novel duck reovirus (NDRV) is a double-stranded RNA virus belonging to the family Reovirdae and the genus Orthoreovirus. To date, two different genotypes of duck reovirus (DRV) have been reported: genotype I and genotype II. Genotype I, Muscovy duck reovirus (MDRV), primarily infects Muscovy ducks and semi-Muscovy ducks, causing necrotizing hepatitis (commonly known as "flowery liver disease"), characterized by numerous millet-like necrotic foci in organs such as the liver and spleen. Genotype II duck reovirus, also known as novel duck reovirus, can infect Peking ducks, Cherry Valley ducks, and Muscovy ducks, causing "splenic necrosis." Infected ducks exhibit white necrosis or hemorrhage on the surface of the spleen, and some livers show punctate necrosis. Compared to MDRV, NDRV has a wider prevalence and poses the greatest threat.

[0003] NDRV has the typical morphology and structure of a reovirus. The virus particles are spherical, with a diameter of about 60nm to 85nm. It is a non-enveloped linear double-stranded RNA virus with a double-capsid icosahedral symmetry structure. The genomic nucleic acid of NDRV is segmented dsRNA. According to electrophoretic mobility results, the virus is composed of three groups of RNA fragments of different sizes. Its genome can be divided into 10 gene segments, including three long segments (L1, L2, L3), three medium segments (M1, M2, M3), and four small segments (S1, S2, S3, S4). Except for the S1 segment, the other genomic segments each have only one open reading frame (ORF) that encodes only one protein. The encoded proteins are also divided into large segment λ proteins (λA, λB, λC), medium segment μ proteins (μA, μB, μNS, μBS, μBN), and small segment σ proteins (σA, σB, σNS, σC, P10, P18). The S1 gene has a polycistronic structure and contains three overlapping ORFs that simultaneously encode σC, p10, and p18 proteins. The L fragment migration rate is similar to that of MDRV, while the M and S fragments differ from MDRV. The S1 gene shows the lowest homology and the greatest difference, exhibiting high variability. NDRV encodes 14 proteins: 10 structural proteins and 4 non-structural proteins. The structural proteins influence viral replication, infection, and transmission. The NDRV S1 gene shows the lowest homology with other reoviruses. The third ORF encodes the σC protein, a major structural protein containing 322 amino acids, which has only 36.3% amino acid homology with the MDRV σC protein. This indicates that alterations in the σC protein gene significantly affect the host range of reovirus infection. The σC protein is located in the viral capsid and, in its normal form, is an oligomeric protein that mediates host cell adhesion, forming homotrimers that interact with receptor molecules. These trimers are bound together by non-covalent interactions and possess surface antigenic determinants that can be recognized by specific neutralizing antibodies, inducing the production of population-specific neutralizing antibodies. Furthermore, studies have shown that increasing the concentration of both monovalent and divalent salts can enhance the stability of the σC protein trimer, and trimer formation also influences the process of viral infection of cells. Meanger et al. suggested that σC protein-mediated syncytial activity is related to the infectivity of NDRV. Therefore, the σC protein plays an important role in viral infection and pathogenicity.

[0004] Monoclonal antibodies are highly homogeneous antibodies produced from a single B cell clone, targeting only a specific antigenic epitope. They can be prepared using hybridoma cell technology, exhibiting high purity, low cost, strong specificity, high efficiency, and no residue, making them suitable for the diagnosis, prevention, and treatment of corresponding diseases. Based on this, this study prepared a novel monoclonal antibody against the σC protein of duck reovirus using hybridoma cell technology and screened for the antigenic epitope located at σC using the monoclonal antibody, providing technical support for the prevention and control of novel duck reoviruses. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel monoclonal antibody against duck reovirus σC protein.

[0006] The technical solution adopted in this invention is: a novel monoclonal antibody against duck reovirus σC protein, wherein the heavy chain of the monoclonal antibody is of type IgG1 and the light chain is of type Kappa.

[0007] Preferably, the monoclonal antibody-positive hybridoma cells are three strains, named 5B7, 3C10, and 6G11.

[0008] Preferably, the positive hybridoma cells for the monoclonal antibody are 6G11.

[0009] Preferably, the smallest epitope of the monoclonal antibody is 304-314aa.

[0010] Preferred epitopes 304 WQDLVPVWLQ 314 The σC protein sequence of duck reovirus is highly conserved.

[0011] The beneficial effects obtained by this invention are as follows: This invention expresses recombinant NDRV σC protein, and obtains a monoclonal antibody with high titer and good activity using hybridoma technology. Using this monoclonal antibody, a novel antigenic epitope of NDRV was screened and identified. 304 WQDLVPVWLQ 314 This epitope is highly conserved in NDRV strains and is a specific antigenic epitope for NDRV. Attached Figure Description

[0012] Figure 1 Amplification diagrams of recombinant plasmids pET-28a-σC and pGEX-4T-1-σC;

[0013] Figure 2 PCR identification of recombinant plasmids;

[0014] Figure 3 SDS-PAGE analysis of recombinant protein expression;

[0015] Figure 4 Image showing the purification and identification of recombinant σC protein;

[0016] Figure 5 Western blot identification of recombinant σC protein;

[0017] Figure 6 Selection of GST-σC recombinant expressed protein and serum dilution;

[0018] Figure 7 To select the optimal action time for serum antibodies;

[0019] Figure 8 For the determination of serum titer in immunized BALB / c mice;

[0020] Figure 9 For monoclonal antibody titer;

[0021] Figure 10 For the analysis of Western blot results;

[0022] Figure 11 For the identification of subclasses of monoclonal antibodies;

[0023] Figure 12 IFA analysis for monoclonal antibodies;

[0024] Figure 13 Purification of ascites fluid;

[0025] Figure 14 Antigenicity analysis of recombinant σC protein;

[0026] Figure 15 Epitope prediction for recombinant σC protein;

[0027] Figure 16 A schematic diagram of the truncated recombinant σC protein;

[0028] Figure 17 Identification of N1-N5 truncated proteins;

[0029] Figure 18 The reactivity of the monoclonal antibody with the truncated peptides N1-N5;

[0030] Figure 19 Identification images for N5-1, N5-2, and N5-3;

[0031] Figure 20 The reactivity of the monoclonal antibody with N5-1, N5-2 and N5-3;

[0032] Figure 21 Identification diagram of the N5-N14 truncated protein;

[0033] Figure 22The reactivity of the monoclonal antibody with the N5-N14 truncated protein;

[0034] Figure 23 Analysis of the conservation of antigenic epitopes;

[0035] Figure 24 This is a representation of the σC protein antigenic epitope in a 3D model. Detailed Implementation

[0036] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0037] A novel monoclonal antibody against duck reovirus σC protein has been developed, wherein the heavy chain of the monoclonal antibody is of the IgG1 type and the light chain is of the Kappa type. Three hybridoma cell lines were identified as positive for the monoclonal antibody and named 5B7, 3C10, and 6G11. The 6G11 cell line was identified as the positive hybridoma cell line for the monoclonal antibody. The smallest epitope of the monoclonal antibody is 304-314aa. 304 WQDLVPVWLQ 314 The σC protein sequence of duck reovirus is highly conserved.

[0039] 1. Materials and Methods

[0040] 1.1 Materials

[0041] 1.1.1 Cells, vectors, and laboratory animals

[0042] The E. coli prokaryotic expression vectors pET-28a and pGEX-4T-1 were both preserved in our laboratory; SP2 / 0 cells were preserved in our laboratory; E. coli competent cells DH5α and BL21 were purchased from Beijing Bomed Gene Technology Co., Ltd.; and the NDRV strain was provided by the Veterinary Biological Products Laboratory.

[0043] 1.1.2 Main Reagents

[0044] Table 1. Main Reagents and Manufacturers

[0045]

[0046] 1.1.3 Main Instruments

[0047] Table 2. Main Instrument Names and Manufacturers

[0048]

[0049] 1.1.4 Preparation of main solutions

[0050] Table 3. Main Reagents and Preparation Methods

[0051]

[0052] 1.2 Preparation of novel duck reovirus σC protein

[0053] 1.2.1 Primer Design

[0054] Based on the σC gene sequence of NDRV in GenBank, primers for amplifying the σC gene were designed, with the upstream primer containing the restriction enzyme site BamHI and the downstream primer containing the restriction enzyme site XhoⅠ (Table 4).

[0055] Table 4 NDRV-σC Amplification Primers

[0056] Primer name Primer sequences (5′-3′) NDRV-σC-F CGCGGATCCGCGatgagacacagctatattcag NDRV-σC-R CCGCTCGAGCGGTTActagagaacggacttgtaac

[0057] 1.2.2 Viral RNA Extraction

[0058] (1) Add four times the volume of Trizol to 250 μL of the amplified virus solution, shake well, let stand in the refrigerator for 5 min, centrifuge at 4℃, 12000 rpm for 5 min, and transfer 800 μL of supernatant into a sterile tube.

[0059] (2) Add 200 μL of chloroform, invert and mix well until it turns milky white. Let stand in an ice box for 5 minutes.

[0060] Centrifuge at 4℃, 12,000 rpm for 15 min.

[0061] (3) Transfer the supernatant to a new centrifuge tube, add pre-cooled isopropanol of equal volume to the supernatant, mix by inversion, place in the refrigerator to stand for 5 min, centrifuge at 4℃, 12000 rpm for 10 min.

[0062] (4) Discard the supernatant, add 1 mL of 75% ethanol to the precipitate, and centrifuge at 4℃ and 8000 rpm for 5 min.

[0063] (5) Discard the supernatant, keep the tube wall dry, add ddH2O, mix by blowing and aspiration, and measure the concentration. Viral RNA extraction is complete.

[0064] (6) Store the extracted RNA in a -80℃ freezer for later use.

[0065] 1.2.3 Construction and amplification of recombinant plasmids

[0066] The extracted sample was reverse transcribed into cDNA. The reverse transcription system is as follows:

[0067] Table 5 Reverse Transcription System

[0068] Components Dosage dNTP Mix 2μL 5×RT Buffer 2μL DTT 1μL Primer Mix 1μL RNase-Free 0.5μL HiFiScript 0.5μL template 3μL total 10μL

[0069] Using this as a template and NDRV-σC-F / NDRV-σC-R as amplification primers, PCR amplification was performed in the following system:

[0070] Table 6 PCR reaction system

[0071]

[0072] PCR reaction procedure: Pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s; annealing at 55℃ for 30 s; extension at 72℃ for 40 s, repeated 30 cycles; final extension at 72℃ for 10 min; storage at 4℃. After the reaction, nucleic acid was identified by agarose gel electrophoresis, and the target gene band was recovered and purified using a gel extraction kit. The recovered product was double-digested with pET-28a / pGEX-4T-1 plasmid using BamHI and XhoⅠ, and reacted at 37℃ for 8 h. Double digestion reaction system:

[0073] Table 7 Enzymatic digestion system of target fragment and vector

[0074]

[0075]

[0076] After the system was completed, the enzyme digestion products were recovered and purified by 1% agarose gel electrophoresis. The digested and purified σC target fragment was then ligated to the vector fragment, and the ligation system is as follows:

[0077] Table 8. Reaction system for linking target fragment to vector

[0078] Element volume T4 DNA Ligase 2μL 10×T4 DNA Ligase Buffer 1μL carrier 2.5μg Target fragment 4.5μL

[0079] After reacting at 22℃ for 3 hours, the ligation product was placed on ice. After the DH5α competent cells thawed, the ligation product was added to the ice. The bottom of the tube was gently tapped to mix the DH5α and the product thoroughly. The mixture was then placed on ice for 30 minutes, followed by heat shock at 42℃ for 50 seconds. After that, the mixture was placed on ice for 2 minutes. 500 μL of antibiotic-free LB liquid medium was added and the mixture was shaken and cultured for 1 hour. The cultured bacterial solution was centrifuged for 5 minutes, 400 μL of LB was discarded, and the bacterial sludge was resuspended using the remaining 100 μL of medium. The bacterial solution was then evenly spread on solid medium with the corresponding resistance (Amp+ / Kana+) in a clean bench without RNase. The mixture was incubated upside down at 37℃ for 12 hours, and the growth of positive colonies was observed.

[0080] Single colonies were picked and cultured in liquid medium with appropriate antibiotics for 3 hours. Preliminary identification was performed using T7 primers via culture PCR. Positive colonies were amplified and plasmids were extracted. The plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, and the results were compared with the NDRV-σC sequence. The correctly identified recombinant plasmid was named pET-28a-σC / pGEX-4T-1-σC.

[0081] 1.2.4 Identification of recombinant proteins

[0082] Transform the correctly identified plasmid into BL21(DE3) competent cells according to step 2.2.3. Pick well-grown single colonies and add them to liquid culture medium containing the corresponding antibiotic at a ratio of 1:100. After culturing for 4-6 hours, take samples for testing. Wait for OD... 600 When the protein concentration reaches 0.4-0.6, IPTG is added, and samples are collected after 4-6 hours of incubation. Expression is verified using SDS-PAGE. The induction culture medium is centrifuged at 8000-9000 rpm for 10 minutes at 4°C, the supernatant is discarded, and the precipitate is resuspended in 12 mL PBS. The collected bacteria are sonicated using an ultrasonic disruptor to fully release the protein. The sonication product is centrifuged at low temperature, and samples are taken for SDS-PAGE analysis to verify whether the recombinant protein is expressed in soluble or inclusion body form. All samples are processed with equal amounts of 4× protein loading buffer.

[0083] 1.2.5 Purification of recombinant proteins

[0084] The pET-28a-σC / pGEX-4T-1-σC cells were resuspended in 12 mL of PBS. The cells were then sonicated for 15 min on ice (intensity 30%, power 360 W, repeated twice). After centrifugation at 12000 rpm for 10 min at 4 °C, the precipitate was collected and dissolved in denaturing buffer containing 8 mol / L urea for 2 h or longer at 4 °C. The precipitate was then centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was filtered through a 0.45 μm filter to remove impurities.

[0085] Purification was performed using the Ni-Agatose Resin method, with the following specific steps:

[0086] (1) Add the mixed Ni-Agatose Resin packing and 20% ethanol to the gravity column, let it stand at room temperature and wait for the layers to separate, discard the ethanol soaking night, add 5 column volumes of deionized water to the column for rinsing, and then add 10 column volumes of Binding Buffer to equilibrate the chromatography column.

[0087] (2) After filtering the protein sample, add it into the column and control the flow rate.

[0088] (3) Rinse the column with Binding Buffer at 15 times the column volume.

[0089] (4) While controlling the flow rate, use Elution Buffer to elute the protein, collect the eluent, and store it at -20°C.

[0090] (5) Wash the chromatography column with Binding Buffer and deionized water, seal the chromatography column with 20% ethanol, and store it at 4-8℃.

[0091] First, add the fusion protein into a dialysis bag and seal both ends with clamps. Dialysis is performed at 4°C, with the refolding solution changed every 4 hours. After dialysis, aspirate the liquid from the dialysis bag and store it at -20°C.

[0092] 1.3 Preparation of novel duck reovirus monoclonal antibody

[0093] 1.3.1 Animal Immunization

[0094] All experimental procedures were approved by the Experimental Animal Ethics Committee of Hebei Agricultural University. His-σC recombinant protein was selected as the antigen for animal immunization and was thoroughly emulsified with Freund's complete adjuvant at a 1:1 volume ratio. Six- to eight-week-old female BALB / c mice were randomly divided into two groups. Mice in the first group received multiple subcutaneous injections of 100 μg of His-σC antigen emulsified with Freund's complete adjuvant in the neck and back. Subsequent immunizations were then performed using Freund's incomplete adjuvant, with each injection being 25 μg. Mice in the second group received a subcutaneous injection of 100 μL PBS as a negative control. Injections were administered every 14 days, and blood was collected one week after the last injection to determine antibody titers. To ensure antibody levels in the mice, a booster immunization of 25 μg of purified His-σC protein was administered intraperitoneally three days before cell fusion.

[0095] 1.3.2 Establishment and optimization of a novel duck reovirus indirect ELISA method

[0096] An antibody detection method using GST-σC as the coating antigen was established to detect antibody titers in immunized mice and to screen hybridoma cells subsequently. This method can exclude antibodies carrying His-tagged proteins used during mouse immunization during the screening and detection process.

[0097] 1.3.2.1 Preliminary Establishment of the Indirect ELISA Method

[0098] (1) Coat a 96-well microplate with 2 μg / mL GST-σC protein, 100 μL / well, overnight at 4°C;

[0099] (2) Remove the coating solution, wash with PBST 3 times, 1 min each time, and pat dry any remaining liquid.

[0100] (3) Block with PBST containing 10% fetal bovine serum for 2 hours, react at 37°C, and wash and dry as above.

[0101] (4) Discard the blocking solution, wash with PBST and pat dry the residual liquid, dilute the mouse positive and negative serum at a ratio of 1:100, 100 μL per well, and incubate at 37°C for 1 h;

[0102] (5) Discard the primary antibody, wash 3 times, 1 min each time, pat dry the remaining liquid, and then dilute goat anti-mouse HRP-IgG at 1:5000 according to the instructions, 100 μL per well, and incubate at 37℃ for 1 h.

[0103] (6) Add two-component TMB solution, 100 μL / well for color development, and react in the dark for 15 min.

[0104] (7) Add 50 μL of 2M sulfuric acid to each well to terminate the reaction. Then, immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0105] 1.3.2.2 Optimization of Indirect ELISA Method Conditions

[0106] GST-σC recombinant protein was used as the coating antigen to coat ELISA plates at concentrations of 1, 2, 4, 8, 16, and 32 μg / mL. 100 μL was added to each well and incubated overnight at 4°C. Positive and negative sera were diluted 1:200, 1:400, 1:800, 1:1600, and 1:3200 to create a checkerboard pattern for determining the optimal antigen coating concentration and the optimal dilution factor for the serum samples. Negative and positive serum samples were tested. The p-value (P / N) ratio was compared and analyzed; the optimal reaction conditions for the ELISA method were determined based on the highest p-value (P / N).

[0107] 1.3.3 Serum antibody titer detection

[0108] Blood was collected from mice after booster immunization and placed at 37°C for 2 hours to allow serum to separate. Serum titer was detected using the ELISA method defined in section 1.3.2, with SPF-grade BALB / c mice serving as a negative control. 450nm The data were observed and compared to analyze the P / N ratio.

[0109] 1.3.4 Hybridoma cell fusion

[0110] 1.3.4.1 Preparation of feeder cells

[0111] Feeder cells were prepared the day before cell fusion. Negative BALB / c mice were euthanized by cervical dislocation after blood was collected from their eyes. The mice were then immersed in 75% alcohol for 5 minutes. After removing the mice and draining excess alcohol, they were fixed in place. The abdominal skin was cut open using a sterile autoclave to expose the peritoneum, which was then disinfected again. The cells were gently lifted with forceps, and 1640 culture medium was injected into the peritoneum multiple times. The abdomen was gently patted and massaged to facilitate the release of peritoneal macrophages. The culture medium was aspirated using a syringe, and this process was repeated several times. The culture medium containing peritoneal macrophages was diluted with complete culture medium, then transferred to centrifuge tubes and centrifuged. The supernatant was discarded, and the cells were resuspended in HAT-containing medium for cell counting. Finally, approximately 40,000 cells per well were added to 100 μL of the medium and cultured in a 96-well plate, avoiding shaking as much as possible.

[0112] 1.3.4.2 Cell Fusion

[0113] Resuscitate SP2 / 0 cells in advance and culture them until they are in good growth condition. Take the best-performing SP2 / 0 cells, discard the supernatant, and resuspend the cells in culture medium into centrifuge tubes for later use.

[0114] Blood samples were collected from the eyes of mice with the highest titers to collect positive serum. The mice were then euthanized by cervical dislocation and disinfected by soaking in alcohol. The mice were then placed on a workbench and secured. The abdominal skin was grasped with sterile forceps, and the outer layer of skin was cut open with sterile scissors, leaving the peritoneum intact. New sterile forceps and scissors were used to cut the peritoneum in the same manner, fully opening the abdominal cavity. The spleen was removed and placed in serum-free 1640 culture medium. Excess tissue around the spleen was removed, and the spleen was placed in a cell strainer. Several small holes were made at one end of the spleen. The spleen was repeatedly aspirated with 1640 cell culture medium and pipetted until it became nearly transparent. The syringe plunger was removed, and the spleen in the cell strainer was repeatedly crushed. The mouse spleen cell suspension was then mixed by pipetting and placed in a 50 mL sterile centrifuge tube and washed three times with serum-free culture medium to remove impurities.

[0115] SP2 / 0 cells and spleen cells were counted separately and then mixed in 15 mL sterile centrifuge tubes at a ratio of 1:3 to 1:8. Centrifuge at 1000 rpm for 12 min. Discard the supernatant and aspirate the culture medium from the tube walls, gently tapping the bottom of the tube to mix the cells. Add 1 mL of preheated PEG4000 fusion medium dropwise over 45 s, rotating the centrifuge tube to ensure effective binding of the fusion medium to the cells on the tube wall. After standing for 90 s, slowly to rapidly add 40 mL of 1640 complete culture medium to the centrifuge tube, and place the cells in an incubator for 10 min to complete the fusion process. Next, centrifuge at low speed to discard the supernatant, and finally add an appropriate amount of HAT-containing medium to resuspend the cells. Plate 100 μL / well of cells into cell culture plates and incubate in a cell culture incubator. Avoid moving the cells for 3 days before fusion. On the fourth day, replace half of the medium with HAT complete medium. In the following days, culture the cells in 10% / 20% HT fetal bovine serum medium according to their growth.

[0116] 1.3.4.3 Screening of hybridoma cells

[0117] Three to four days after fusion, small cell clusters can be observed forming under a microscope. At this time, macrophages and unfused cells gradually detach and die. By eight to eleven days after fusion, hybridoma cells begin to grow at the bottom of the wells, covering approximately one-quarter or one-third of the well bottom area. At this point, the cell supernatant is screened using the indirect ELISA method established in section 1.3.2.

[0118] 1.3.5 Subcloning

[0119] Hybridoma cell clusters that tested positive in both tests were subcloned. One day in advance, feeder cells were prepared according to section 1.3.4.1 and plated. After dispersing the cell clusters with HT-containing medium, the cells were counted. Approximately 100 cells were selected and diluted. The diluted solution was evenly distributed into 96-well plates containing feeder cells for culture. During culture, the number and growth status of cells in each well were observed and recorded regularly. When the cells grew to large colonies, the supernatant was analyzed using indirect ELISA to screen for positive and well-growing cells, which were then further screened. After four rounds of screening and identification, the cells were passaged. For cells that continuously produced antibodies, three cryopreservation and thawing procedures were performed to ensure their stability.

[0120] 1.3.6 Preparation of Monoclonal Antibodies

[0121] Pregnant BALB / c female mice were selected. Seven days prior to the experiment, 0.6 mL of sterile liquid paraffin was administered intraperitoneally to stimulate peritoneal cells. Subsequently, the revived positive hybridoma cells were diluted and counted at 10 per mouse. 6 / Injected into sensitized mice via intraperitoneal injection. When the mice are observed to have swollen abdomens and slow movement, ascites can be collected. Ascites is extracted from the mice's abdomen using a syringe, centrifuged for 20 to 30 minutes, then aliquoted and stored at -80°C with added glycerol.

[0122] 1.3.7 Characterization of Monoclonal Antibodies

[0123] 1.3.7.1 Monoclonal antibody titer determination

[0124] The indirect ELISA method established in section 2.3.2 was used to detect antibody titers in ascites fluid. First, ascites fluid samples were serially diluted 1:400. Ascites fluid induced by SP2 / 0 cells was used as a negative control. This method allows for accurate assessment of antibody titers in ascites fluid samples.

[0125] 1.3.7.2 Identification of Monoclonal Antibody Subtypes

[0126] Following the instructions for the antibody subclass identification kit produced by Suzhou Bio-Long Technology Co., Ltd., the obtained monoclonal antibodies were identified as follows:

[0127] (1) Allow the solution to return to room temperature for 30 minutes before use. Dilute the cleaning solution with deionized water to the required working concentration.

[0128] (2) Set the number of sample wells on the ELISA plate: 8 wells for each sample and 8 wells for each positive and negative control.

[0129] (3) Pipette 50 μL of sample diluent into the detection well and add 50 μL of cell supernatant containing antibody. Mix the two by pipetting and aspirating. Add 100 μL directly to the positive and negative controls and incubate at 37°C for 1 h.

[0130] (4) Discard the liquid in the plate, wash thoroughly with washing solution and pat dry the liquid in the wells, then add the matching enzyme-labeled secondary antibody to the detection wells, 100 μL per well, and incubate at 37°C for 1 h.

[0131] (5) Discard the liquid in the plate, wash thoroughly with cleaning solution and pat dry the liquid in the well. Add 50 μL each of the well-mixed substrate solution A and solution B to the detection well and incubate in the dark for 15 min.

[0132] (6) Use an ELISA reader at 450 nm to measure the values ​​to determine the subtype of the monoclonal antibody.

[0133] 1.3.7.3 Western blot analysis of monoclonal antibodies

[0134] (1) After thoroughly mixing the monoclonal antibody sample with the protein loading buffer, boil for 10 minutes;

[0135] (2) Add 10-15 μL of monoclonal antibody sample and marker to the gel well and run the gel;

[0136] (3) Cut a PVDF membrane and protein glue to the appropriate size and place them in the middle of the transfer filter paper. Transfer the membrane in an ice bath for 2 hours.

[0137] (4) Block with TBST containing 5% skim milk powder for 2 hours, rinse clean, add primary antibody, and incubate with shaking at room temperature for 1 hour;

[0138] (5) At room temperature, the sample was first washed with TBST buffer, then the secondary antibody was added and incubated for 1 h. After that, the sample was washed three times with PBST buffer for 5 min each time.

[0139] (6) Finally, place the strip in a chemiluminescence imager, add chemiluminescence reagent (ECL), and acquire an image.

[0140] 1.3.7.4 Indirect Immunofluorescence Identification with Monoclonal Antibodies

[0141] First, seed Vero cells in 48-well plates until the cell density reaches approximately 80%. Then, inoculate Vero cells with NDRV virus and culture for 48-72 hours, after which discard the culture medium. Next, fix with pre-chilled paraformaldehyde for 20-30 minutes. Wash thoroughly with PBS and gently blot dry the liquid in the wells. Then, add 100 μL of 0.1% Triton X-100 to each well for permeabilization; wash and blot dry as before, then block with 5% BSA in PBS for 1 hour. After washing, add 400 μL of diluted monoclonal antibody (1:200 concentration) to each well and incubate at 37°C for 1 hour. After washing the cells, add 100 μL of FITC-labeled secondary antibody to each well and react at 37°C for 1 hour. Wash three times with PBS under dark conditions. After washing, do not completely drain the liquid; leave enough PBS to cover the cells to prevent cell desiccation. Finally, observe the results under an inverted fluorescence microscope.

[0142] 1.3.8 Purification of Monoclonal Antibodies

[0143] First, crude extraction is performed using the octanoic acid-saturated ammonium sulfate precipitation method, as follows:

[0144] (1) Mix the ascites fluid with PBS at a ratio of 1:2, and then add 3 mL of ammonium sulfate and stir.

[0145] (2) Centrifuge the mixture at 8000 rpm for 15 min at 4℃ and collect the precipitate;

[0146] (3) Resuspend the precipitate in PBS, dialyze in 200 mL of PBS, and incubate overnight at 4°C;

[0147] After initial purification, further purification was performed using G protein agar affinity chromatography, as follows:

[0148] Pack the protein G chromatography medium into the column and equilibrate with 5 column volumes of buffer to ensure the packing material is in the same buffer system as the target protein. Add the sample to the column and collect the eluent. Wash the column with 10-15 column volumes of eluent and collect the eluent containing the target protein. Adjust the pH of the target protein fraction to neutral using neutralization buffer. Equilibrate the packing material sequentially with 3 column volumes of binding buffer and 5 column volumes of deionized water, and finally equilibrate with 5 column volumes of 20% ethanol. Store the purified eluent in an equal volume of 20% ethanol at 4°C. Analyze the purification effect using SDS-PAGE compared to the unpurified ascites fluid.

[0149] 1.4 Identification of antigenic epitopes of novel duck reovirus

[0150] 1.4.1 Prediction of antigenic epitopes

[0151] This study used DNASTAR software and the Immunotope Database (IEDB) resource website to predict and analyze the sequence characteristics and potential antigenicity of the σC protein in the NDRV S1 gene. The locations of possible antigenic epitopes were identified, followed by a detailed analysis of these predicted epitopes, and finally, a corresponding truncation strategy was developed.

[0152] 1.4.2 Primer Design and Synthesis

[0153] To identify the linear B-cell epitope located in NDRVσC, specific primers were designed based on the NDRVσC gene sequence (Table 9), and restriction enzyme sites BamHI and XhoⅠ were inserted. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0154] Table 9. Shortened peptide primers for antigen epitope identification

[0155]

[0156]

[0157] 1.4.3 Construction and Expression of Truncated Proteins

[0158] In constructing the truncated protein vector, specific primers are first designed to amplify the truncated protein. Next, the amplified product is ligated to the expression vector pGEX-4T-1 via double enzyme digestion. After PCR identification, the recombinant plasmid is sent to a sequencing institution for sequence analysis. Once the sequencing results are confirmed to be correct, it is transformed into BL21 competent cells for induction. Finally, the expressed truncated protein is identified by Western blot using a GST-tagged antibody for further validation.

[0159] 1.4.4 Preliminary identification of antigenic epitopes

[0160] In the initial epitope localization, the full-length NDRVσC protein gene was first designed into five truncated fragments, N1-N5, which were then reacted with monoclonal antibodies. Based on the experimental results, the truncated proteins were progressively shortened to precisely locate the antigenic epitopes. During this process, the GST-tagged truncated proteins were used as antigens for peptide ELISA and dot blot assays to identify the antigenic epitopes recognized by the monoclonal antibodies.

[0161] The procedure for peptide ELISA is as follows:

[0162] (1) Using GST-σC protein as a positive control and empty vector as a negative control, the truncated protein was diluted to 4 μg / mL with CBS buffer, 100 μL per well, and coated on the microplate overnight at 4℃.

[0163] (2) Discard the coating solution and wash the plate three times with PBST solution. Then, use PBS containing 5% fetal bovine serum as the blocking solution, add 200 μL to each well, and incubate at 37°C for 2 h;

[0164] (3) Discard the blocking solution, wash the plate thoroughly with PBST and pat dry any remaining liquid. Use 100 μL of monoclonal antibody diluted 1:800 per well and incubate at 37°C for 1 h.

[0165] (4) Discard the liquid in the well, wash 3 times, and use 1:5000 diluted goat anti-mouse HRP-IgG, 100 μL / well, and incubate at 37℃ for 1 h;

[0166] (5) Discard the liquid in the well, wash and dry, add substrate TMB solution, 100 μL / well, and incubate at 37°C for 15 min in the dark.

[0167] (6) Add 50 μL of 2M sulfuric acid solution to each well to terminate the reaction, and then immediately use an ELISA reader to measure the absorbance (OD) value of each well at a wavelength of 450 nm.

[0168] The specific steps for performing a Dot blot operation are as follows:

[0169] First, a suitable amount of PVDF membrane was cut and activated with methanol for 3 min. The sample was centrifuged at 4°C, and the supernatant was used as the secreted protein. The PVDF membrane was then air-dried until semi-dry. 3 μg of sample, positive control (GST-σC), and negative control (empty vector) were respectively placed on the PVDF membrane and air-dried until the sample was absorbed by the membrane and the color deepened. Then, the membrane was blocked with TBST solution containing 5% skim milk and incubated at 37°C for 2 h or overnight. Subsequently, the monoclonal antibody was diluted 1:200 with TBST solution containing 5% skim milk and incubated at 37°C for 1 h. Next, the PVDF membrane was thoroughly washed with TBST solution. Then, it was incubated with enzyme-labeled secondary antibody (1:5000 dilution) at 37°C for 1 h. Finally, the PVDF membrane was thoroughly washed again with TBST solution and exposed to ECL luminescence solution.

[0170] 1.4.5 Conservation Analysis of Antigenic Epitopes

[0171] We searched GenBank for representative reovirus strains, compared the identified epitope sequences with the amino acids at the corresponding σC positions in these sequences, and analyzed the conservation of the antigenic epitopes recognized by the monoclonal antibodies identified in this study.

[0172] 1.4.6 Biological Analysis

[0173] The three-dimensional structure of the NDRVσC protein was constructed by simulating its amino acid sequence, and the location of the antigenic epitope in the three-dimensional structure was analyzed.

[0174] 2 Results and Analysis

[0175] 2.1 Preparation of novel duck reovirus σC protein

[0176] 2.1.1 Identification of recombinant plasmids

[0177] Using the extracted NDRV gene as a template, specific primers were designed to amplify the full-length NDRVσC gene, obtaining a 966bp target gene band that matched the size of the σC gene. Figure 1 The target gene was cloned into vectors pET-28a and pGEX-4T-1, and then transformed into competent DH5α cells. Subsequent double enzyme digestion identification revealed vector bands of 5300 bp and 4969 bp, and the target band of 966 bp, respectively. Figure 2 The sequencing results, compared with NCBI, confirmed the sequence as the NDRVσC gene sequence. These results indicate that the recombinant plasmids pET-28a-σC and pGEX-4T-1-σC were successfully obtained.

[0178] Figure 1A: M: DM2000 Marker; 1: pET-28a-σC amplification result;

[0179] B: M: DM2000 Marker; 1: pGEX-4T-1-σC amplification results.

[0180] Figure 2 A: M: DL5000 DNA Marker; 1: pET-28a-σC enzyme digestion product;

[0181] B: M: DL5000 DNA Marker; 1: pGEX-4T-1-σC digestion product.

[0182] 2.1.2 Expression and Identification of Recombinant Proteins

[0183] The obtained recombinant plasmids were transformed into competent cells for induced expression, and named His-σC and GST-σC, respectively. SDS-PAGE analysis showed that the His-σC recombinant protein expressed a target protein of approximately 35 kDa after induction, while the GST-σC recombinant protein expressed a target protein of approximately 46 kDa, consistent with expectations. The solubility of the recombinant proteins was analyzed by SDS-PAGE of the solubility supernatant and precipitate after sonication. The results showed... Figure 3 The target protein was almost entirely present in the precipitate of the ultrasound product, existing in the form of inclusion bodies. Subsequently, inclusion body proteins were selected for further experiments and protein purification.

[0184] Figure 3 In Chinese: A: M: Protein Marker; 1: Before His-σC induction; 2: After His-σC induction; 3: Supernatant after induction; 4: Precipitate after induction. B: M: Protein Marker; 1: Before GST-σC induction; 2: After GST-σC induction; 3: Supernatant after induction; 4: Precipitate after induction.

[0185] 2.1.3 Purification and identification of novel duck reovirus σC protein

[0186] After purification of His-σC and GST-σC proteins using affinity chromatography, SDS-PAGE electrophoresis analysis revealed a single band without obvious impurities, and the purified protein's molecular weight was consistent with the expected target protein. Figure 4 This indicates that the protein purification effect was good.

[0187] Figure 4 In Chinese: A: His-σC recombinant protein; B: GST-σC recombinant protein.

[0188] The immunogenicity of the recombinant proteins was identified by Western blot. His-σC and GST-σC recombinant proteins were reacted with His- and GST-tagged antibodies, respectively. The results are shown in the figure. Figure 5 Both heavy proteins showed clear, specific bands.

[0189] Figure 5 In Chinese: A: His-σC recombinant protein; B: GST-σC recombinant protein.

[0190] 2.2 Establishment of Indirect ELISA Detection Method

[0191] 2.2.1 Determination of optimal antigen coating concentration and serum dilution

[0192] According to the results of the matrix titration experiment, under the conditions of GST-σC as the coating antigen concentration of 4 μg / mL and serum dilution of 1:400, the P / N value reached a maximum of 7.405. The results are as follows... Figure 6 As shown, the optimal antigen coating concentration and the dilution of the serum to be tested were determined accordingly.

[0193] 2.2.2 Determination of the optimal incubation time for the serum to be tested

[0194] The optimal reaction time for the serum samples was optimized, and the results are as follows: Figure 7 As shown, when the serum to be tested is incubated for 60 minutes, the P / N value can reach a maximum of 6.99, confirming that the optimal incubation time for the serum to be tested is 60 minutes.

[0195] 2.3 Determination of titer in immunized mice

[0196] Blood was collected from the tail tip of mice after immunization and the titer was determined using an established indirect ELISA method. Serum from negative mice was used as a control. The results showed that ( Figure 8 The serum of mice 1-5 were diluted to 1:128000 and all showed positive results, meeting the conditions for cell fusion. Among them, mouse 1 had the highest titer and was the preferred choice for hybridoma cell fusion experiments.

[0197] 2.4 Preparation of Monoclonal Antibodies

[0198] The growth status of cells after fusion was closely observed under a microscope. Cells that failed to fuse and feeder cells died successively. 10-12 days after fusion, obvious cell colonies could be observed in successfully fused cells. The cell clusters were screened using an established indirect ELISA to obtain three positive hybridoma cell lines that could secrete monoclonal antibodies against NDRVσC, which were named 5B7, 3C10, and 6G11.

[0199] 2.4.1 Determination of Monoclonal Antibody Titer

[0200] The prepared monoclonal antibody was serially diluted in ascites fluid and detected by indirect ELISA. The results showed that ( Figure 9 The titers of the three monoclonal antibodies all ranged from 5.12 × 10⁻⁶. 4 Up to 2.048×10 5 The titers of 6G11 in ascites fluid were as high as 1:409600, while the lowest titer of 3C10 was still 1:204800. These results indicate that these three monoclonal antibodies possess good titers and reactivity.

[0201] 2.4.2 Identification of the reactivity of monoclonal antibodies

[0202] To further detect the reactivity of monoclonal antibodies 5B7, 3C10, and 6G11, Western blot analysis was performed using GST-σC recombinant protein as the antigen and ascites fluid as the primary antibody. Results showed that ( Figure 10 ), 5B7, 3C10 and 6G11 can produce specific bands against GST-σC protein, indicating that the prepared monoclonal antibodies can specifically bind to σC protein and have good reactivity.

[0203] 2.4.3 Identification of Monoclonal Antibody Subtypes

[0204] According to the instructions of the antibody identification kit, the subclasses of the three monoclonal antibodies were identified. The results are shown in Figure (11). The heavy chains of monoclonal antibodies 5B7, 3C10 and 6G11 are all of type IgG1, and their light chains are all of type κ.

[0205] 2.4.4 Indirect immunofluorescence identification

[0206] Vero cells were infected with NDRV strains. When obvious lesions were observed under a microscope, monoclonal antibodies 5B7, 3C10, and 6G11 were used as primary antibodies, and SP2 / 0 cell supernatant was used as a negative antibody control. NDRV-infected Vero cells showed specific green fluorescence after incubation with secondary antibodies, while the negative control group did not. The results showed ( Figure 12 No specific green fluorescence was observed in 3C10, while monoclonal antibodies against strains 5B7 and 6G11 specifically recognized the σC protein of NDRV and showed good reactivity.

[0207] 2.4.5 Ascites purification

[0208] Based on the above results, monoclonal antibody 6G11 was selected for further identification and analysis. After purification by ascites fluid, 6G11 was identified by SDS-PAGE, as shown in the figure. Figure 13The purified 6G11 showed heavy and light chains corresponding to mouse-specific IgG at 55kDa and 25kDa, respectively, indicating good purification effect and high purity, and can be used for subsequent experiments.

[0209] Identification of 2.5σC protein antigenic epitopes

[0210] 2.5.1 Prediction of antigenic epitopes

[0211] To locate the antigenic epitope of the NDRVσC protein, its characteristics and epitope regions were analyzed using DNAStar. The results showed (see...). Figure 14 σC protein possesses good hydrophilicity and antigenicity. Based on a comprehensive analysis of the predicted epitopes (see...),... Figure 15 Preliminary screening of full-length σC proteins was conducted.

[0212] 2.5.2 Truncated expression and screening of antigenic epitopes

[0213] This study performed a series of truncations on the NDRVσC protein ( Figure 16 First, five truncated peptides (N1-N5) were constructed, covering the entire sequence of the σC protein, with overlapping fragments at both ends. Western blot analysis showed... Figure 17 These truncated peptides N1-N5 were successfully expressed and recognized by the anti-GST tag antibody. Next, the epitopes were initially screened using the monoclonal antibody 6G11. ELISA and Dot blot results showed that... Figure 18 The monoclonal antibody 6G11 showed a specific response only to the N5 truncated peptide, indicating that its recognition region is located in the N5 truncated peptide.

[0214] Based on the above experimental results, the N5 truncated peptide was selected for further shortening. Three overlapping truncated peptides, N5-1 (235-268 aa), N5-2 (258-304 aa), and N5-3 (271-322 aa), were designed based on N5. Western blot analysis confirmed the successful expression of the three fragments. Figure 20 Based on the results of peptide ELISA and Dot blot assays ( Figure 20 The N5-3 fragment was specifically identified. Further analysis showed that the monoclonal antibody failed to recognize the truncated N5-2 fragment, further localizing the antigenic epitope recognized by the monoclonal antibody 6G11 to the range of 304-322aa.

[0215] Figure 18 In the diagram: A: Peptide ELISA identifies the truncated protein; B: Dot blot identifies the truncated protein.

[0216] Figure 19 In the N5-1, N5-2, and N5-3 identification diagrams: A: PCR identification of N5-1, N5-2, and N5-3; B: Western blot identification of N5-1, N5-2, and N5-3.

[0217] Figure 20 In the reactivity of monoclonal antibodies with N5-1, N5-2, and N5-3: A: Peptide ELISA identification; B: Dot blot identification.

[0218] Finally, to determine the smaller epitopes recognized by the monoclonal antibody 6G11, this study truncated the epitopes every two amino acids from the N-terminus and C-terminus. Western blot analysis revealed the results as follows: Figure 21 As shown, N5-N14 was successfully expressed, based on the results of peptide ELISA and Dotblot assays. Figure 22 The truncated N5-N10 proteins can all be recognized by the monoclonal antibody 6G11, while the truncated N11-N14 proteins do not specifically react with the monoclonal antibody, indicating that 304-314aa is the minimal epitope recognized by the monoclonal antibody.

[0219] Figure 21 The identification diagram of the N5-N14 truncated protein: A: N5-N14 PCR identification; B: N5-N14 Western blot identification.

[0220] Figure 22 In the reactivity of monoclonal antibodies with N5-N14 truncated proteins: A: peptide ELISA identification; B: dot blot identification.

[0221] 2.5.3 Analysis of the conservation of antigenic epitopes

[0222] To assess the conservation of the selected antigenic epitopes, NDRV, GRV, and ARV from different regions were selected from NCBI and compared using MEGA software. The comparison results showed that ( Figure 23 ), indicating the epitope 304 WQDLVPVWLQ 314 It is highly conserved in the NDRVσC protein sequence.

[0223] 2.5.4 Representation of antigenic epitopes in a three-dimensional structural model

[0224] Homology modeling of viral particle structures formed by the σC protein was performed using the SWISS-MODEL website, and then the antigenic epitopes were located using Pymol software. The final results are as follows: Figure 24 As shown in Figure A, the three-dimensional structure of NDRV virus particles can be observed on its surface. 304 WQDLVPVWLQ 314 Epitopes may exist on the surface of protein structures. Figure 24B shows the secondary structure of the σC protein, and speculates... 304 WQDLVPVWLQ 314 Epitopes may exist in a linear manner on the surface of protein structures.

[0225] Figure 24 Representation of σC protein antigenic epitopes in a 3D model: A: 3D model of viral particles; B: Secondary structure of σC protein.

[0226] 3. Conclusion

[0227] (1) Recombinant NDRV σC protein was expressed, and a monoclonal antibody with high titer and good activity was obtained using hybridoma technology. A novel antigenic epitope of NDRV was screened and identified using the monoclonal antibody. 304 WQDLVPVWLQ 314 This epitope is highly conserved in NDRV strains and is a specific antigenic epitope for NDRV.

[0228] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A novel monoclonal antibody against duck reovirus σC protein, characterized in that: The heavy chain of the monoclonal antibody is of type IgG1, and the light chain is of type Kappa.

2. The novel duck reovirus σC protein monoclonal antibody according to claim 1, characterized in that: The monoclonal antibody-positive hybridoma cell lines consist of three strains, which are named 5B7, 3C10, and 6G11.

3. The novel duck reovirus σC protein monoclonal antibody according to claim 1, characterized in that: The positive hybridoma cells for the monoclonal antibody were 6G11.

4. The novel duck reovirus σC protein monoclonal antibody according to claim 3, characterized in that: The smallest epitope of the monoclonal antibody is 304-314aa.

5. The novel duck reovirus σC protein monoclonal antibody according to claim 4, characterized in that: Epitope 304 WQDLVPVWLQ 314 The σC protein sequence of duck reovirus is highly conserved.

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