Duck reovirus σC protein monoclonal antibody and hybridoma cell line and application thereof

Abstract

The present invention discloses a monoclonal antibody against duck reovirus σC protein and its hybridoma cell line and application, belonging to the technical field of biological immunology. The present invention immunizes BALB / c mice with recombinant immune σC protein of purified duck reovirus DRV-TH11 strain, and screens hybridoma cells that can stably secrete duck reovirus σC protein monoclonal antibodies through cell fusion technology to obtain duck reovirus σC protein monoclonal antibodies; by sequencing the hybridoma cells, the light chain and heavy chain sequences of the duck reovirus σC protein monoclonal antibodies are obtained, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4. The antibody prepared by the present invention has high specificity and stability, can specifically identify cells infected with duck reovirus, and is suitable for immunological detection.

Description

Technical Field

[0001] The invention belongs to the technical field of biological immunology, and particularly relates to a duck reovirus σC protein monoclonal antibody and a hybridoma cell line and application thereof. Background Art

[0002] Duck reovirus (DRV) is a member of the avian reovirus genus Orthoreovirus in the family Reoviridae.

[0003] Duck reovirus belongs to the Reoviridae family, Orthoreovirus genus. It is an enveloped, double-stranded RNA segmented virus with an icosahedral symmetry. The virion diameter is approximately 60-80 nm, and the genome is approximately 23.4 kb in length. Based on the size of the double-stranded RNA segments of the viral genome, it is divided into ten segments: L (L1, L2, L3), M (M1, M2, M3), and S (S1, S2, S3, S4). In addition to the S1 segment, which contains three cistrons, namely three open reading frames encoding the p10, p18, and σC proteins, the remaining nine segments contain only a single cistron, each encoding a single protein. The σC protein is the primary capsid protein of DRV, involved in viral adsorption and proliferation, promoting syncytium formation, and inducing the production of specific and protective neutralizing antibodies. Therefore, the σC protein has become the preferred antigen for the development of new genetically engineered vaccines and diagnostic reagents for duck reovirus.

[0004] Currently, the detection technologies for duck reovirus include virus isolation, RT-PCR, RT-qPCR, indirect immunofluorescence (IFA), ELISA and other methods, but the detection takes a long time and is expensive, and there is currently no commercial kit for the detection of duck reovirus. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a duck reovirus σC protein monoclonal antibody and its hybridoma cell line and application, so as to solve the problems of long detection time and high cost of duck reovirus detection in the prior art.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a monoclonal antibody against the duck reovirus σC protein is provided, wherein the heavy chain variable region of the monoclonal antibody has three complementarity determining regions (VH CDR1, VH CDR2 and VH CDR3), which are composed of the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; and the light chain variable region has three complementarity determining regions (VLCDR1, VL CDR2 and VL CDR3), which are composed of the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0007] The beneficial effects of the present invention are as follows: the present invention immunizes BALB / c mice with the recombinant immune σC protein of the purified duck reovirus DRV-TH11 strain, screens out a hybridoma cell that can stably secrete a duck reovirus σC protein monoclonal antibody through cell fusion technology, and obtains the duck reovirus σC protein monoclonal antibody; and obtains the light chain and heavy chain sequences of the duck reovirus σC protein monoclonal antibody by sequencing the hybridoma cells.

[0008] On the basis of the above technical solution, the present invention can also be improved as follows:

[0009] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0010] Furthermore, the subtype of the monoclonal antibody against duck reovirus σC protein was IgG2.

[0011] Another object of the present invention is to provide a nucleic acid molecule that can encode the above-mentioned anti-duck reovirus σC protein monoclonal antibody.

[0012] Another object of the present invention is to provide an expression vector comprising the above nucleic acid molecule.

[0013] Another object of the present invention is to provide a hybridoma cell line that secretes the monoclonal antibody against duck reovirus σC protein as claimed in the claims, wherein the hybridoma cell line is mouse hybridoma cell 1D5E8, whose preservation number is CGMCCNO.45840, and which was deposited in the General Microbiology Center of China Culture Collection Administration on April 10, 2024.

[0014] Another object of the present invention is to provide the use of the above-mentioned duck reovirus σC protein monoclonal antibody in the preparation of a kit for detecting duck reovirus.

[0015] Furthermore, the kit is a kit for Western Blot detection.

[0016] Furthermore, the kit is an indirect immunofluorescence IFA detection kit.

[0017] Furthermore, the kit is an immunohistochemistry IHC detection kit.

[0018] The present invention has the following beneficial effects: the present invention provides a hybridoma cell line that can secrete duck reovirus σC protein monoclonal antibody, the antibody has high specificity and stability, can specifically identify cells infected with duck reovirus, and is suitable for immunological detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of Western blotting for detecting the recognition specificity of the monoclonal antibody in Example 3, wherein M is the standard molecular weight pre-stained protein, A is the detection result of BHK-21 cells infected with DRV, and B is the detection result of the negative BHK-21 cell control;

[0020] Figure 2 The results of the indirect immunofluorescence detection of the monoclonal antibody recognition specificity in Example 4, wherein A is the detection result of BHK-21 cells infected with DRV, and B is the detection result of the negative BHK-21 cell control;

[0021] Figure 3 This is the lung staining result of the negative control sample of immunohistochemical staining in Example 5;

[0022] Figure 4 This is the result of DRV-infected lung staining in Example 5. DETAILED DESCRIPTION

[0023] The principles and features of the present invention are described below in conjunction with the examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0024] Example 1: Preparation of monoclonal antibodies against duck reovirus σC protein

[0025] 1. Virus seeds and cells

[0026] Virus species: duck reovirus DRV-TH11 strain.

[0027] Cells: BHK-21 cells and SP2 / 0 cells were preserved by the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0028] 2. Construction of recombinant plasmid for prokaryotic expression of σC protein

[0029] Viral RNA was extracted from the DRV-TH11 strain virus liquid using the Trizol extraction method, and the obtained RNA was reverse transcribed into cDNA, which was used as a template for the PCR system to amplify the DRVσC gene and cloned into the prokaryotic expression plasmid pET-30a.

[0030] The successfully constructed recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) strain, and the recombinant His-σC protein was expressed by IPTG induction.

[0031] The recombinant σC protein was purified using a nickel column, and the protein concentration was determined by BCA.

[0032] 3. Immunization of BALB / c mice

[0033] Select 6-8 week old female BALB / c mice. Purified σC protein was emulsified with an equal volume of Freund's complete adjuvant (Sigma, catalog number F5881) and injected intraperitoneally and subcutaneously at multiple points on the back of the BALB / c mice at a dose of 50-100 μg. Two and four weeks after the initial immunization, purified σC protein was emulsified with an equal volume of Freund's incomplete adjuvant (Sigma, catalog number F5506) for the second and third immunizations, respectively. One week after the final immunization, blood was collected and serum antibody titers were measured using an indirect ELISA. Cell fusion was performed when the ELISA titer reached 1:105 or higher. Three days before fusion, a booster immunization was performed with an intraperitoneal injection of 100 μg of the unadjuvanted antigen solution.

[0034] 4. Preparation of feeder cells

[0035] One day before cell fusion, blood was collected from the eyeballs of unimmunized BALB / c mice (8-12 weeks old), and the mice were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes. The mice were placed in a porcelain dish on a clean bench and fixed on a foam board with the abdomen facing up. The skin of the BALB / c mice was lifted with sterilized tweezers, and the abdominal skin was carefully cut open to separate the skin and peritoneum to fully expose the peritoneum. An appropriate amount of DMEM nutrient solution was injected into the abdominal cavity with a sterile syringe. The abdomen was massaged. After the peritoneal cells and the injected nutrient solution were fully mixed, the nutrient solution was gently sucked back into a sterile centrifuge tube. The resulting solution was centrifuged at 1000 r / m for 5 minutes, and the supernatant was discarded. 10 mL of DMEM nutrient solution was added to the cell pellet, the cells were re-blown, and the cells were resuspended in DMEM culture medium containing HAT (Sigma, catalog number H0262) to adjust the cell concentration to 1-2 × 10 5 100 μL / well was added to a 96-well cell culture plate and placed in a 37° C., 5% CO 2 incubator for culture until ready for use.

[0036] 5. Culture of myeloma cells (SP2 / 0)

[0037] One day before fusion, the SP2 / 0 cells were divided into flasks and cultured in 75 cm 2 On the day of fusion, select SP2 / 0 cells with good morphology and logarithmic growth, gently blow them off the flask wall, collect them in a 50 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes for cell fusion.

[0038] 6. Preparation of immune spleen cells

[0039] On the third day after the booster immunization, the immunized mice were taken, their eyeballs were removed and bled, and the serum was separated as a positive control for antibody detection; the mice were killed by cervical dislocation and soaked in 75% alcohol for 5 minutes and placed on a porcelain plate on a clean bench; the mouse spleen was aseptically removed and placed in a plate containing 10 mL of DMEM nutrient solution, and gently rinsed to remove attached connective tissue and fat; the spleen was placed on a 70-mesh cell sieve, ground with a 2 mL syringe inner core, and rinsed with DMEM nutrient solution so that all spleen cells passed through the mesh and entered the solution; the spleen cell solution was transferred to a 50 mL centrifuge tube, DMEM nutrient solution was added to 30 mL, mixed, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded; the cells were centrifuged and washed once in the same way, and then the cells were suspended in 10 mL of DMEM nutrient solution and mixed.

[0040] 7. Cell Fusion

[0041] Spleen cells from immunized mice were mixed with SP2 / 0 cells in a ratio of 5-10:1 in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the bottom of the tube was gently tapped to loosen and evenly disperse the precipitated cells into a paste. 1 mL of 50% PEG (WT 1450, Sigma, catalog number P5402) (pH 8.0) solution preheated to 37°C was pipetted with a 1 mL pipette and slowly dripped along the rotating tube wall, controlling the addition to be completed within 60 s. The mixture was allowed to stand for 60 s, and 25 mL of DMEM nutrient solution was added to the centrifuge tube within 5 min to terminate the fusion reaction. The fused cell solution was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in HAT culture medium preheated in a 37°C water bath. The cells were added to a 96-well culture plate containing feeder cells at 100 μL / well and cultured in a 37°C, 5% CO2 incubator. Cell growth was recorded every day. On the fourth day after fusion, half of the culture medium was replaced with 1% HT selection medium. About 7-10 days later, the culture medium was fully replaced with 1% HT (Sigma, catalog number H0137) selection medium and culture was continued.

[0042] 8. Screening and cloning of positive hybridoma cells

[0043] Observe cell growth daily. 3-5 days after fusion, visible clones will emerge. When colonies reach 1 / 3-1 / 2 of the bottom of the well, test the supernatant using an indirect ELISA. For cells in wells positive for specific antibodies, clone them three times using limiting dilution to achieve a 100% positive rate. Promptly freeze the cells, then expand the culture to prepare ascites fluid. Serially passage, freeze, and resuscitate a portion of the cells to observe the stability of the monoclonal antibody secreted by the cells.

[0044] After indirect ELISA screening and three subcloning steps, a hybridoma cell line that stably secretes a monoclonal antibody against duck reovirus σC protein was obtained. This hybridoma cell line has been deposited. The following is the detailed information of the deposit:

[0045] Classification: Mouse hybridoma cells;

[0046] Name: 1D5E8;

[0047] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0048] Abbreviation of depository institution: CGMCC;

[0049] Address of the depository institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0050] Deposit number: CGMC NO.45840;

[0051] Date of preservation: April 10, 2024.

[0052] This cell line has the biological properties of both parents. After intraperitoneal inoculation into BALB / c mice, it can form tumors and stably produce specific antibodies. After stable passaging for 3 months, it was frozen in liquid nitrogen. After 3 and 6 months of recovery, the cells grew well and the antibody secretion level did not decrease.

[0053] 9. Preparation of Monoclonal Antibody Ascites

[0054] The hybridoma cell line that can stably secrete duck reovirus σC protein monoclonal antibody was expanded and cultured, and the monoclonal antibody was prepared by inducing ascites in syngeneic mice.

[0055] BALB / c mice aged 10 weeks or older were selected and each was intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Seven days later, hybridoma cells (1×105 cells / mouse) were intraperitoneally inoculated. Starting from the seventh day after intraperitoneal injection of hybridoma cells, the abdomen of the mouse was observed daily. When the abdomen of the mouse was significantly enlarged, the peritoneal cavity was punctured with a No. 9 needle and ascites was collected. The ascites was centrifuged at 2000 rpm for 10 minutes to remove oil and precipitate. The supernatant was collected, the antibody titer was determined, and the supernatant was stored at -20°C until further use to obtain monoclonal antibodies (ascites).

[0056] 10. Purification of monoclonal antibodies

[0057] The monoclonal antibody (ascites) obtained in step 9 was centrifuged at 12,000 rpm for 30 min, and the supernatant was mixed with an equal volume of PBS buffer (pH 7.4). The protein was separated and purified using a Protein A+G Agarose prepacked column (Shanghai Biyuntian Biotechnology Co., Ltd.). The protein eluate was collected to obtain the purified monoclonal antibody (ascites). The concentration was determined using a BCA protein assay kit, and the purified protein was aliquoted and stored at -20°C for future use.

[0058] 11. Cloning of the gene encoding the monoclonal antibody against reovirus σC protein

[0059] The anti-reovirus σC protein monoclonal antibody hybridoma cells obtained in step 8 were collected, RNA was extracted by Trizol method and reverse transcribed into cDNA, the monoclonal antibody heavy chain and light chain genes were amplified by PCR technology, cloned into pCE2-TA vector, and the sequence was confirmed by sequencing.

[0060] As shown in Table 1, the gene sequence of the light chain variable region (VL) of the monoclonal antibody is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the gene sequence of the heavy chain variable region (VH) is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0061] Table 1 Monoclonal antibody gene sequence and amino acid sequence

[0062] Heavy chain variable region gene sequence (5'-3', 354 bp) SEQ ID NO.1 Heavy chain variable region amino acid sequence (118aa) SEQ ID NO.2 Light chain variable region gene sequence (5'-3', 321 bp) SEQ ID NO.3 Light chain variable region amino acid sequence (107aa) SEQ ID NO.4

[0063] SEQ ID NO.1:

[0064] GAGGTTCAGC TGCAGCAGTC TGGACCTGAG CTGGTGAAGC CTGGGGCCTC AGTGAAGATA 60

[0065] TCCTGCAAGG CTTCTGGTTA CTCATTTACT GGCTACTTTA TGAACTGGGT GAAGCAGAGC 120

[0066] CATGGAAAGA GCCTTGAGTG GATTGGACGT ATTCATCCTT ACAATGGTGA TACTTTCTAC 180

[0067] AACCAGAAGT TCAAGGGCAA GGCCACATTG ACTGTAGACA AATCCTCTAG CACAGCCCAC 240

[0068] ATGGAGCTCC TGAGCCTGAC ATCTGAGGAC TCTGCAGTCT ATTATTGTGG AAGAGGGGGT 300

[0069] AACGACTGGT ACCTCGATGT CTGGGGCGCA GGGACCACGG TCACCGTCTC CTCA 354

[0070] SEQ ID NO.2:

[0071] EVQLQQSGPELVKPGASVKISCKASGYSFTGYFMNWVKQSHGKSLEWIGRIHPYNGDTFYNQKFKGKATLTVDKSSSTAHMELLSLTSEDSAVYYCGRGGNDWYLDVWGAGTTVTVSS

[0072] SEQ ID NO.3:

[0073] GACATCCAGA TGACTCAGTC TCCAGCCTCC CTATCTGCAT CTGTGGGAGA AACTGTCACC 60

[0074] ATCACATGTC GAGCAAGTGG GAATATTCAC AAATATTTAG CATGGTTTCA GCAGAAACAG 120

[0075] GGAAAATCTC CTCAGCTCCT GGTCTATAAT GCAAAAACCT TAGCAGATGG TGTGCCATCA 180

[0076] AGGTTCAGTG GCAGTGGATC AGGAACACAA TATTCTCTCA AGATCAACAG CCTGCAGCCT 240

[0077] GAAGATTTTG GGAGTTATTA CTGTCAACAT TTTTGGAGTA CTCCGTGGAC GTTCGGTGGA 300

[0078] GGCACCAAGC TGGAAATCAG A 321

[0079] SEQ ID NO.4:

[0080] DIQMTQSPASSLSASVGETVTITCRASGNIHKYLAWFQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIR

[0081] The heavy chain variable region (VH) has three complementarity determining regions (CDRs): VH CDR1, which consists of the amino acid sequence shown in SEQ ID NO. 5; VH CDR2, which consists of the amino acid sequence shown in SEQ ID NO. 6; and VH CDR3, which consists of the amino acid sequence shown in SEQ ID NO. 7.

[0082] The light chain variable region (VL) has three complementarity determining regions (CDRs), namely: VL CDR1, which consists of the amino acid sequence shown in SEQ ID NO.8; VL CDR2, which consists of the amino acid sequence shown in SEQ ID NO.9; and VL CDR3, which consists of the amino acid sequence shown in SEQ ID NO.10.

[0083] The specific amino acid sequence is shown in Table 2:

[0084] Table 2 Amino acid sequences of each CDR region of the heavy chain and light chain

[0085]

[0086]

[0087] Example 2: Determination of biological properties of monoclonal antibodies

[0088] 1. Identification of antibody classes and subclasses

[0089] The monoclonal antibody subclass identification kit (Beijing Biolong Immunotechnology Co., Ltd.) was used to identify the subclasses of the monoclonal antibodies of the hybridoma cell line of duck reovirus σC protein monoclonal antibodies according to the instructions of the kit.

[0090] The results showed that the heavy chain of the monoclonal antibody secreted by the hybridoma cell line was IgG2 and the light chain was kappa chain.

[0091] 2. ELISA titer determination

[0092] The purified DRV-TH11 strain virus recombinant σC protein obtained in Example 1 was diluted to 1 μg / mL with carbonate buffer (pH=9.6) and coated with 100 μL / well of an ELISA plate at 4°C overnight. Block with PBST containing 5% skim milk (PBS containing 0.05% Tween 20) at 37°C for 2h; wash three times with PBST solution, add 10-fold serial dilutions (diluent is PBS) of the monoclonal antibody (cell supernatant) obtained in step 8 of Example 1 and the monoclonal antibody (ascites) obtained in step 9, 100 μL / well, and incubate at 37°C for 1h; wash five times with PBST solution, add 100 μL / well of horseradish peroxidase-labeled anti-mouse secondary antibody (CST company, 1:5000 dilution) and incubate at 37°C for 1h; wash five times with PBST solution, add 100 μL / well of TMB color development solution, incubate at 37°C for 15min; stop the reaction by adding 50 μL of 2MH2SO4 to each well, shake gently to mix, and measure the absorbance of each well (OD) with a microplate reader. 450nm value), with the OD of the experimental group 450nm / OD of the control group 450nm The wells with a P / N > 2.1 were judged as positive (i.e., the highest dilution factor when P / N > 2.1 was the endpoint for titer determination).

[0093] SP2 / 0 cell culture supernatant and SP2 / 0 cell ascites were prepared using the method in Example 1 as negative controls.

[0094] The results are shown in Table 1. The ELISA antibody titer of the monoclonal antibody cell supernatant and ascites obtained from the hybridoma cell line number and DRV-TH11 recombinant σC protein was 1:10. 4 and 1:10 7 .

[0095] Table 3 OD detection by indirect ELISA of cell culture supernatant and ascites 450nm value

[0096]

[0097] Example 3

[0098] 1. Application of Duck Reovirus σC Protein Monoclonal Antibody in Western Blot Detection

[0099] BHK-21 cells not infected with DRV were used as negative control, and BHK-21 cells infected with DRV were used as test samples.

[0100] The cells were lysed with RIPA lysis buffer, 5×SDS protein loading buffer was added, and the cells were heated at 100°C for 10 min. SDS-PAGE gel electrophoresis was performed and the cells were transferred to a cellulose acetate membrane (NC) for protein immunoblot analysis. The primary antibody was the monoclonal antibody obtained in step 8 of Example 1 (cell culture supernatant). The secondary antibody was a horseradish peroxidase-labeled anti-mouse secondary antibody (CST, 1:10,000 dilution).

[0101] 2. Results Analysis

[0102] See the results Figure 1 As shown, M: standard molecular weight pre-stained protein; A: DRV-TH11 strain infected BHK-21 cell protein; B: BHK-21 cell control protein. It can be seen that the monoclonal antibody (cell culture supernatant) and the DRV-TH11 strain viral protein produce a clear and single band at approximately 37Ku. The size of this band is consistent with the size of the DRV-TH11σC protein, and it has no reaction with the BHK-21 cell control protein.

[0103] Example 4

[0104] 1. Application of Duck Reovirus σC Protein Monoclonal Antibody in IFA Detection Technology

[0105] BHK-21 cells not infected with DRV were used as negative control, and BHK-21 cells infected with DRV were used as test samples.

[0106] The cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature and washed three times with PBS. They were permeabilized with 0.3% Triton X-100 (diluted in PBS) for 10 minutes at room temperature and washed three times with PBS. The cells were blocked with 5% BSA (diluted in PBS) at 37°C for 2 hours and washed three times with PBS. The primary antibody was the monoclonal antibody (cell culture supernatant) obtained in step 8 of Example 1 above. The cells were incubated at room temperature for 1 hour and washed three to five times with PBST. The secondary antibody was Texas Red-labeled goat anti-mouse IgG (Invitrogen, 1:2000 dilution). The cells were incubated at room temperature for 1 hour and washed three to five times with PBST. DAPI (Shanghai Biotech Co., Ltd., 1:3000 dilution) was incubated for 5 minutes at room temperature.

[0107] 2. Results Analysis

[0108] See the results Figure 2 As shown, A: DRV-TH11 strain infected BHK-21 cell protein; B: BHK-21 cell control. It can be seen that the monoclonal antibody (cell culture supernatant) reacts with BHK-21 cells infected with DRV-TH11 strain and emits red fluorescence, but has no reaction with the BHK-21 cell control protein and no red fluorescence is observed.

[0109] Example 5

[0110] 1. Application of Duck Reovirus σC Protein Monoclonal Antibody in IHC Staining

[0111] 1. Antigen retrieval: 0.01M sodium citrate retrieval solution (pH=6.0) with high pressure heat retrieval;

[0112] 2. Inactivation of endogenous peroxidase: Wash the sections with PBS buffer three times for 5 minutes each time to remove the buffer from the sections; immerse the sections completely in 3% hydrogen peroxide solution and incubate at room temperature for 10 minutes;

[0113] 3. Blocking: Wash the sections three times with PBS buffer for 5 minutes each time to remove the buffer from the sections. Use an immunohistochemistry pen to circle the tissue area to be examined on the slide and add 5% BSA blocking solution to the circled area. Place the sections horizontally in a wet incubation chamber with water at the bottom and incubate at room temperature for 30 minutes, starting from the time the blocking solution was added.

[0114] 4. Primary antibody incubation: Remove the blocking solution, add the primary antibody diluted with antibody solution to the tissue section, place it horizontally in an incubation humidified chamber, and incubate at room temperature for 60 minutes; remove the antibody working solution, quickly rinse once with PBS buffer, and soak and wash three times with PBS buffer, each time for 5 minutes; during the soaking and washing period, the sections need to be repeatedly lifted up and down (primary antibody dilution ratio 1:5000);

[0115] 5. Secondary antibody incubation: Add horseradish peroxidase-labeled anti-mouse secondary antibody (CST, 1:5000 dilution) to the tissue sections and place them horizontally in a humidified incubator for 45 minutes at room temperature. Remove the reagent from the sections, quickly rinse once with PBS buffer, and then soak and wash three times with PBS buffer, each for 5 minutes. During the soaking and washing period, lift the sections up and down repeatedly.

[0116] 6. Color development: Add DAB color developing solution to the tissue section, closely observe the color change under a microscope, and after obtaining the appropriate staining intensity, immerse the section in a large amount of distilled water to stop the color development. After stopping the color development, wash the section in running water for 10 minutes.

[0117] 7. Restaining: Immerse the slightly drained sections in hematoxylin solution for 10 minutes. After restaining, rinse with running water for 10 minutes.

[0118] 8. Bluing: Immerse the slightly drained slices in a saturated aqueous solution of lithium carbonate for 3 seconds, then rinse with running water for 3 minutes;

[0119] 9. Dehydration: Soak the cleaned slices in anhydrous ethanol once. During the soaking period, lift them up and down several times. After 10 seconds, take them out. Place them in a constant temperature blast drying oven at high temperature (54-58°C) to completely dry.

[0120] 10. Sealing: Add an appropriate amount of neutral gum to the center of the slice and cover it with a cover slip. The amount of glue added should be appropriate. After adding the cover slip, the tissue should be completely covered without any glue overflowing.

[0121] 11. Slice scanning.

[0122] 2. Results Analysis

[0123] Immunohistochemical staining results are divided into positive and negative, and positive expression must be in specific antigen sites of cells and tissues to be considered positive.

[0124] Using anti-duck reovirus monoclonal antibodies, specific staining was observed in samples infected with duck reovirus; lungs in negative control samples were negative; the specific results are as follows Figure 3 and Figure 4 As shown, the results show that the anti-duck reovirus σC protein monoclonal antibody staining is accurately positioned, with clear staining and no nonspecific staining, and a clean background. Combined with the results of immunoblotting and immunofluorescence, it shows that the anti-duck reovirus monoclonal antibody specifically recognizes the duck reovirus σC protein, without nonspecific staining, and can effectively avoid false positive results.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A monoclonal antibody against duck reovirus σC protein, characterized in that: The heavy chain variable region of the monoclonal antibody has three complementarity determining regions, VH CDR1, VH CDR2 and VH CDR3, which are composed of the amino acid sequences shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively; the light chain variable region has three complementarity determining regions, VL CDR1, VL CDR2 and VL CDR3, which are composed of the amino acid sequences shown in SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10, respectively.

2. The duck reovirus σC protein monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. The duck reovirus σC protein monoclonal antibody according to claim 2, characterized in that Its subtype is IgG2.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule is a nucleic acid molecule encoding the anti-duck reovirus σC protein monoclonal antibody according to any one of claims 1 to 3.

5. An expression vector, characterized in that The vector comprises the nucleic acid molecule of claim 4.

6. A hybridoma cell line secreting the duck reovirus σC protein monoclonal antibody according to any one of claims 1 to 3, characterized in that: The hybridoma cell line is mouse hybridoma cell 1D5E8, whose preservation number is CGMCCNO.45840, and it was deposited in the General Microbiology Center of China Culture Collection Administration on April 10, 2024.

7. Use of the monoclonal antibody against duck reovirus σC protein according to any one of claims 1 to 3 in the preparation of a kit for detecting duck reovirus.

8. The use according to claim 7, characterized in that The kit is a kit for Western Blot detection.

9. The use according to claim 7, characterized in that The kit is an indirect immunofluorescence IFA detection kit.

10. The use according to claim 7, characterized in that The kit is an immunohistochemistry (IHC) detection kit.

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