LU503352B1 - A method for identifying antigenic epitopes of the óC protein of the novel aquatic reovirus - Google Patents

Abstract

The present invention relates to the technical field of identifying antigen expressions in aquatic birds, in particular to a method for identifying antigenic epitopes of the σC protein of the novel aquatic reovirus, with the advantageous effect that using indirect immunofluorescence results all four strains of MAbs were positive for novel duck reovirus (NDRV) and negative for classic duck reovirus (CDRV) and avian duck reovirus (ARV), the antigenic epitopes recognized by the four strains of MAbs were determined using synthetic overlapping peptide libraries and a series of truncated peptides, and the results showed that the central antigenic epitope recognized by the three monoclonal antibodies A5-A1, A5-B6 and B9-6 was 177PILSGPADA185 and the antigenic epitope, recognized by the A9-D4 monoclonal antibody was likely in a spatial conformation, and antigenic epitope conservativity analysis showed that the antigenic epitopes were highly conserved in NDRV isolates from different geographic regions and different host sources, demonstrating fundamental Provides information for further study of the structure of the SigC protein and development of clinical tests for NDRV.

Description

A method for identifying antigenic epitopes of the novel hr/>03352 cC protein

waterfowl reovirus

Technical part

The present invention relates to the technical field of identification of

Antigenic expression in aquatic birds, particularly to a method for identifying antigenic epitopes of the 6C protein of the novel aquatic reovirus.

technology in the background

Duck reovirus (DRV) is divided into two genotypes, type I and type

II. Genotype I is the Classical Duck Reovirus (CDRV), which primarily infects ducks, proboscis ducks and geese, and causes necrotizing hepatitis (commonly known as "duck spotting" or "flower liver disease" in ducks). , the

Disease was introduced to China in the 1990s and subsequently became epidemic; genotype II is a novel duck reovirus (Novel DRV,

NDRYV), which causes disease in a variety of waterfowl characterized by severe

necrosis and hemorrhage in the liver and spleen (commonly known as "hemorrhagic necrotizing hepatitis"), the disease was first detected in China in the early 21st century, and the causative agent has been isolated and identified. Currently, genotype II (NDRV) is the predominant genotype in China.

The NDRV genome consists of 10 segments of double-stranded RNA and the virus particle consists of an icosahedrally symmetrical double envelope with a diameter of 70 nm without a capsid. The genome can be divided into three large fragments (L1-3), three medium fragments (M1-3) and four small fragments (S1-4), which can encode at least 10 structural and four non-structural proteins, such as the Show SDS-PAGE electrophoresis results. The SigC protein is encoded by the 3rd ORF of the S1 segment and is a small component of the virus's outer coat. The SigC protein has been shown to associate with surface antigens for virus-type specific neutralization responses, thereby stimulating the body to produce protective neutralizing antibodies; in addition, the protein is located on the surface of the outer envelope and can recognize and attach to target cells through receptor-mediated recognition, thus functioning as a receptor. In addition, SigC proteins can induce apoptosis in host cells via a p53-dependent pathway.

SigC is the most variable of the proteins encoded by avian duck reovirus, NDRV, CDRV and ARV all belong to the avian orthorectal duck reovirus population, but the NDRV

SigC protein shares low amino acid homology with the SigC proteins of CDRV and

ARV up, about 40% and 30% respectively. NDRYV is currently the predominant genotype and there are no antigenic B cell epitopes for NDRV. In addition, the epitopic antigenicity of the NDRV

SigC protein and the existence of type-specific antigenic epitopes unknown.

content of the invention

The aim of the present invention is to provide a method for identifying antigens

Novel aquatic reovirus cC protein epitopes to provide epitopes to the above

to solve the problems addressed by the prior art.

To achieve this, the invention offers the following technical solutions:

A method for identifying antigenic epitopes of the cC protein of the novel

Aquatic fowl reovirus, the method of identification comprising the following steps:

S1: Preparation of the raw material

Raw materials: cells, virus voice and experimental animals, virus voice including NDRV

ZJOOM and CDRV ZJ2000M, cells including chicken embryo filbroblast DF-1; LU503352

S2: Preparation of the reagents

Reagents: pET-28a-his vector, test kit, HRP-labeled sheep anti-mouse IgG (HRP-

IgG), Forster's Complete Adjuvant, Forster's Incomplete Adjuvant, PEG4000,

Hypoxanthine Thymine Nucleoside, Hypoxanthine Methotrexate Thymine Nucleoside,

S3: Construction, protein expression and purification of the recombinant plasmid pET-28a-

SignC

According to the S1 gene sequence of the NDRV ZJOOM strain, the Oligo6.0

Software designed a primer pair that uses the SigC ORF of the S1 gene as a target region, and

NDRYV ZJOOM was taken to inoculate DF-1 cells, 200 µl of cell lysate was added after 72

Hours taken, RNA extraction was performed, and the target fragment SigC was amplified, and the target fragment SigC was obtained by amplification and pET-28a-His vector to obtain recombinant plasmid pET-28a-SigC;

The SigC of the recombinant plasmid pET-28a-SigC and the vector pET-28a-His were subjected to NcoI/XhoI double digestion, the digested products were ligated and transformed, and the resulting recombinant plasmid pET-28a-SigC was double digested and sequencing verified, the validated recombinant plasmid pET-28a-SigC was transformed into receptor cells and expression was induced by isopropyl-p-D-thiogalactopyranoside (1.0 mol/L) for 5 h, and centrifuge to collect the bacteria and perform sonication , and supernatants and principals were separated;

Analysis of recombinant protein expression and purification of recombinant proteins, resulting in determination of protein concentration and analysis of its purity by the analyzer;

S4: immunization of animals

immunization of purified recombinant SigC protein in culture;

SS: Cell fusion and production of MAbs

Spleen cells from mice were compared with myeloma cells from

Mice fused and positive hybridoma cells capable of stably secreting anti-SigC protein-specific MAbss were assayed by ELISA, and for the assayed positive hybridoma cells stably secreting antibodies, ascites was assayed by conventional

Methods established, and the effectiveness of the ascites antibody was determined by indirect

ELISA detected;

S6: Identification of the biological characteristics of MAbs (1) Identification of MAbs subtypes

Identification of the prepared ascites antibodies with the MAbs subtype

identification kit; (2) Western blot analysis of MAbs

Collection of DF-1 cells infected with NDRV, visualization and analysis of the

Western blot bands by kit controls; (3) Indirect immunofluorescence identification

performing control experiments to determine the binding activity of MAbs to NDRV and to assess whether MAbs have binding activity to CDRV and ARV;

S7: Identification and localization of antigenic epitopes by peptide scanning

A library of 32 SigC-1-32 peptides with a truncated length of 15 amino acids and an overlap of 5 amino acids of the SigC protein was artificially synthesized, and each peptide was dissolved with DMSO and coated at 1 µg/well, and the ELISA -Screenirg/ 503352 was probed with the prepared MAbs as primary antibody and HRP-labeled sheep anti-

mouse IgG as a secondary antibody, and the prepared anti-SigC protein

Mouse serum with high immunity was used as a positive control and mouse serum without immunity as a negative control for the preliminary identification of antigenic epitopes by indirect

ELISA used;

The 32 SigC protein peptides (SigC-1-32) were truncated with the peptides found in the MAbs

were strongly positive in ELISA, and the synthesis of 8 SigC-18-1-8 peptides was continued by stepwise truncation from the N- and C-terminal ends, respectively, with a cis shift of 2 amino groups each, and the 8 truncated peptides were combined with coated ELISA plates and the antigenic epitope sequence was determined from the OD4s0 value using the same ELISA method as above;

S8: Analysis of Conservatism

Avian reoviruses such as NDRV and CDRV from different generations and geographic locations were selected and their respective SigC protein amino acid sequences analyzed and compared to examine the conservativity of the antigenic epitopes.

Preferably, in step 2, the kits include: DL15000 DNA markers, MiniBEST Viral

RNA/DNA Extraction Kit Ver.5.0, PrimeScriptTM One Step RT-PCR Kit, Agarose Gel Recovery

Kit, NcoI and XhoI restriction endonucleases and plasmid extraction kit, MAb isoform

Detection Kit, ECL Chemiluminescence Detection Kit, Ni-NTA Kit.

Preferably, the starting materials in step 1 include, in particular, mouse

myeloma cells SP2/0, ARV S1133 strain, E. coli DH5a and BL21 receptor cells, and 6-8

Week-old BALB/c mice.

Preferably, in step 3, the target fragment SigC is extracted using the

RNA amplified as template using the PrimeScriptTM One Step RT-PCR Kit, PCR

Reaction system: PrimeScript One Step Enzyme Mix 1 pL, 2xOne Step Buffer 12.5 uL, 20 pmol/L

Upstream and downstream primers 10 pmol each, RNA template 1.5 pL supplemented with RNase Free dH20 25.0 µL, with one-step RT-PCR reaction conditions: reverse transcription at 50°C for 30 min; predenaturation at 94°C for 2 min; Denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 60 s, a total of 30 cycles were performed.

Preferably, in step 3, the expression of the recombinant protein is analyzed by SDS-PAGE, the recombinant protein SUMO-SigC is purified using a Ni-NTA kit, then the protein concentration is determined using a Quawell Q3000 Nucleic

Acid Protein Analyzer and its purity was analyzed by SDS-PAGE.

Preferably, the processing method for screening by the ELISA

Procedure in step 5 follows: An indirect ELISA method was established using purified recombinant SigC protein as the encapsulated antigen, the

Culture supernatant from the fused hybridoma cells was tested using the established ELISA method, with positive sera from mice with anti-NDRV SigC protein as a positive control and

Sera from non-immunized mice were used as a negative control, hybridoma cells corresponding to the ELISA positive supernatant were collected and assayed by the conventional limiting dilution method, and a total of three were isolated

Rounds of subcloning performed.

The control experiment in step 6 preferably proceeds as follows: after the lysis, the

The supernatant is subjected to decoction and centrifugation for SDS-PAGE electrophoresis, then the PAGE gel is transferred to a nitrocellulose membrane, the N&V508852

Membrane is sealed with 5% skim milk, and the prepared MAbs as primary antibody and goat anti-mouse IgG-HRP as secondary antibody, while DF-1 cells not using

NDRV infected were used as a negative control.

Preferably, in step 4, the immunization culture method comprises the following:

Immunization of 6-8 week old BALB/c mice (100 µg/each) with purified recombinant SigC protein by multipoint subcutaneous injection in the neck and

Booster once at an interval of 2 weeks, a total of three times, 7 days after the third immunization Mice with the highest serum antibody potency (100 µg/piece) were again immunized with SigC protein (without adjuvant), and spleen cells were immunized on the third

Tag fused with SP2/0 cells.

Preferably, in step 6, the method of indirect fluorescence identification comprises:

NDRV ZJOOM was inoculated with DF-1 cells at an infectious dose of 0.01 MOI, and

Cells not inoculated with the virus were used as a negative control and 48

incubated for hours at 37°C and 5% CO2; the medium was discarded and the cells were fixed with 4% paraformaldehyde and identified by indirect immunofluorescence using the prepared MAbs as the primary antibody and goat anti-mouse IgG-FITC as the secondary antibody to determine the binding activity of the MAbs to NDRV.

Preferably, in step 6, DF-1 cells were infected with CDRV ZJ2000M or ARV S1133 at 0.01 MOI, respectively, and cells not inoculated with virus were used as negative controls, incubated for 48 hours, and then identified by IFA to determine whether the MAbs have binding activity for CDRV and ARV, and the western blot bands were visualized and analyzed with the ECL chemiluminescence kit.

Compared with the prior art, the present invention has the following advantages

Effects:

The four MAbs strains specifically recognized the natural SigC protein of NDRV using the peptide scan method to identify and localize antigenic epitopes, the results of indirect immunofluorescence showed that all four MAbs were positive for NDRV and negative for the classic Duck reovirus and avian duck reovirus were the most antigenic

Epitopes recognized by the four strains of MAbs were identified using synthetic overlapping peptide libraries and a series of truncated peptides, and the results showed that the central antigenic epitope recognized by the three monoclonal antibodies AS-A1, A5- B6 and B9-6 was recognized, 177PILSGPADA was 185, and the antigenic epitope recognized by the monoclonal antibody A9-D4 was probably in a spatial conformation, and the conservativity analysis of the antigenic

Epitopes showed that the antigenic epitopes in NDRV isolates from different geographical

Regions and different host sources were highly conserved, providing fundamental information for further studies on the structure of the SigC protein and the

Development of clinical tests for NDRV supplies.

Description of the accompanying drawings

Figure 1 shows a table identifying antibody isoforms derived from the

hybridoma cell line of the present invention;

Figure 2 shows a western blot identification reaction graph for the MAbs of the present invention;

Figure 3 shows an identification response diagram A for the antigenic epitopes of the present invention; LU503352

Figure 4 shows an identification reaction diagram B for the antigenic epitopes of the present invention.

Detailed Description 5 The technical solutions in the embodiments of the present invention are described in

In the following, in conjunction with the accompanying drawings, the embodiments of the present invention are clearly and fully described, and it is clear that the described

Embodiments are only part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments achieved by a person skilled in the art without any creative work fall within the scope of the present invention.

Referring to Figures 1 to 4, the present invention shows a technical

Solution:

A method for identifying antigenic epitopes of the cC protein of the novel

Aquatic fowl reovirus, the method of identification comprising the following steps:

S1: Preparation of the raw material

Raw materials: cells, virus voice and experimental animals, virus strains including NDRV

ZJ00M and CDRV ZJ2000M, cells including chicken embryo filbroblast DF-1 that

In particular, starting materials include mouse myeloma cells SP2/0, the ARV S1133 strain,

E. coli DH5a and BL21 receptor cells and 6-8 week old BALB/c mice;

S2: Preparation of the reagents

Reagents: pET-28a-his vector, test kit, HRP-labeled sheep anti-mouse IgG (HRP-

IgG), Förster's complete adjuvant, Förster's incomplete adjuvant, PEG4000,

Hypoxanthine Thymine Nucleoside, Hypoxanthine Methotrexate Thymine Nucleoside, Kits: DL15000

DNA Markers, MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0, PrimeScriptTM One Step RT-

PCR Kit, Agarose Gel Recovery Kit, Ncol and XhoI Restriction Endonucleases and

Plasmid Extraction Kit, MAb Isoform Detection Kit, ECL Chemiluminescence Detection Kit, Ni-

NTA Kit;

S3: Construction, protein expression and purification of the recombinant plasmid pET-28a-

SignC

According to the S1 gene sequence of the NDRV ZJOOM strain, the Oligo6.0

Software designed a primer pair that uses the SigC ORF of the S1 gene as a target region, the

Target fragment SigC using the extracted RNA as template with the PrimeScriptTM

One Step RT-PCR Kit, PCR reaction system: PrimeScript One Step Enzyme

Mix 1 pL, 2xOne Step Buffer 12.5 pL, 20 pmol/L upstream and downstream primer 10 pmol each,

RNA Template 1.5 µl supplemented with RNase Free dH20 25.0 pL using One-Step RT-PCR

Reaction conditions: reverse transcription at 50°C for 30 min; predenaturation at 94°C for 2 min; Denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 60 s, a total of 30 cycles were performed and NDRV ZJOOM was taken to infect DF-1

To inoculate cells, 200 µl of cell lysate was taken after 72 hours, RNA extraction was performed, and the target fragment SigC was amplified, and the target fragment SigC was obtained by amplification and pET-28a-His vector to obtain the recombinant plasmid pET-28a -

obtain SigC;

The SigC of the recombinant plasmid pET-28a-SigC and the vector pET-28a-His were subjected to NcoI/XhoI double digestion, the digested products were ligated and transformed, and the resulting recombinant plasmid pET-28a-SigC became durd503352

Double digestion and sequencing verified, the validated recombinant plasmid pET-28a-

SigC was transformed into receptor cells, and the expression was induced by isopropyl-B-D-thiogalactopyranoside (1.0 mol/L) for 5 h, and centrifuge to collect the bacteria and perform sonication, and supernatants and precipitates were separated;

The expression of the recombinant protein is analyzed by SDS-PAGE, the recombinant protein SUMO-SigC is purified using a Ni-NTA kit, then the

Protein concentration is determined using a Quawell Q3000 Nucleic Acid Protein Analyzer and its purity was analyzed by SDS-PAGE.

S4: immunization of animals

Immunization of purified recombinant SigC protein in culture, the

Immunization culture procedure includes the following: immunization of 6-8 week olds

BALB/c mice (100 µg/piece) with purified recombinant SigC protein by subcutaneous injection

Multipoint injection in the neck and booster once at an interval of 2

weeks, a total of three times, 7 days after the third immunization mice with the highest

Serum antibody potency (100 µg/piece) were reimmunized with SigC protein (without adjuvant) and spleen cells were fused with SP2/0 cells on the third day;

SS: Cell fusion and production of MAbs

Spleen cells from mice were compared with myeloma cells from

mice, the processing procedure includes for screening by the ELISA

Procedure as follows: An indirect ELISA method was established using purified recombinant SigC protein as the encapsulated antigen, the culture supernatant of the fused hybridoma cells was tested using the established ELISA method, yielding positive

Sera from mice with anti-NDRV SigC protein as a positive control and sera from non-immunized mice as a negative control were used, hybridoma cells conforming to the im

ELISA positive supernatant were removed and compared with the conventional

The limited dilution method was studied and a total of three rounds of the

subcloning performed and positive hybridoma cells capable of expressing anti-SigC

Stable secreting protein-specific MAbss were examined, and for the examined positive hybridoma cells stably secreting antibodies, ascites was treated by conventional ones

methods, and the effectiveness of the ascites antibody was determined by indirect

ELISA detected;

S6: Identification of the biological characteristics of MAbs (1) Identification of MAbs subtypes

Identification of the prepared ascites antibodies with the MAbs subtype

identification kit; (2) Western blot analysis of MAbs

NDRV-infected DF-1 cells were collected and the specific method of

Control experiment was as follows: after lysis, decoction and centrifugation, the

Subject supernatant for SDS-PAGE electrophoresis, then run the PAGE gel on a

Transferred to nitrocellulose membrane, the NC membrane is sealed with 5% skim milk, and the prepared MAbs as primary antibodies and goat anti-mouse IgG-HRP as secondary antibodies, while DF-1 cells not infected with NDRV as Negative controls were used and the western blot bands were visualized and analyzed with the kit. (3) Indirect immunofluorescence identification

NDRV ZJOOM was inoculated with DF-1 cells at an infectious dose of 0.01 MOI, and 503352

Cells not inoculated with the virus were used as a negative control and 48

incubated for hours at 37°C and 5% CO2; the medium was discarded and the cells were fixed with 4% paraformaldehyde and identified by indirect immunofluorescence using the prepared MAbs as the primary antibody and goat anti-mouse IgG-FITC as the secondary antibody to determine the binding activity of the MAbs to NDRV,

DF-1 cells were infected with CDRV ZJ2000M or ARV S1133 at 0.01 MOI, and cells not inoculated with virus were used as negative controls, incubated for 48 hours, and then identified by IFA to determine if the MAbs had a binding activity for CDRV and ARV, and the western blot bands were labeled with the ECL

Chemiluminescence Visualization and Analysis Kit, visualization and analysis to determine binding activity of MAbs to NDRV and to assess whether MAbs have binding activity to CDRV and ARV;

S7: Identification and localization of antigenic epitopes by peptide scanning

A library of 32 SigC-1-32 peptides with a truncated length of 15 amino acids and an overlap of 5 amino acids of the SigC protein was artificially synthesized, and each peptide was dissolved with DMSO and coated at 1 µg/well, and the ELISA -Screening was performed using the prepared MAbs as the primary antibody and HRP-labeled sheep anti-

mouse IgG as a secondary antibody, and the prepared anti-SigC protein

Mouse serum with high immunity was used as a positive control and mouse serum without immunity as a negative control for the preliminary identification of antigenic epitopes by indirect

ELISA used;

The 32 SigC protein peptides (SigC-1-32) were truncated with the peptides found in the MAbs

were strongly positive in ELISA, and the synthesis of 8 SigC-18-1-8 peptides was continued by stepwise truncation from the N- and C-terminal ends, respectively, with a cis shift of 2 amino groups each, and the 8 truncated peptides were combined with coated ELISA plates and the antigenic epitope sequence was determined from the OD4s0 value using the same ELISA method as above;

S8: Analysis of Conservatism

Avian reoviruses such as NDRV and CDRV from different generations and geographic locations were selected and their respective SigC protein amino acid sequences analyzed and compared to examine the conservativity of the antigenic epitopes.

Analysis of the results:

Analysis 1: Prokaryotic expression, identification and purification of the SigC protein

The recombinant plasmid pET-28a-SigC was identified by double digestion with NcoI and XhoI to obtain two fragments matching the expected size of 5236 and 966 bp and the sequencing results showed that the sequences were correct.

E. coli BL21 cells were transformed with pET-28a-SigC and induced by IPTG at 37°C for 5 h, the supernatant and precipitate were collected by ultrasonic lysis of the bacteria and then subjected to SDS-PAGE. The results showed that the expressed recombinant protein was mainly in the form of inclusion bodies in the cell precipitate with a molecular weight of about 35 kD, as expected. Western blot assay of the purified recombinant SigC protein showed a specific reaction band at 35 kD indicating good reactivity of the expression product with the anti-allovirus serum;

Analysis 2: Identification of MAbs subtypes and determination of ascites effect

After cell fusion, the resulting hybridoma cells were indirect ELISAY503352

method and the positive hybridoma cells were subclonally purified by the three-fold limiting dilution method to obtain four positive hybridoma cell lines stably secreting anti-NDRV SigC protein MAbs and named A5-A1, A5-B6,

Wear A9-D4 or B9-6. The results of monoclonal antibody isotype testing showed that all four hybridoma cell lines secreted IgG1 antibodies and the light chains of the antibodies secreted were all x (as shown in Figure 1). 4 hybridoma cell supernatants, A5-

A1, A5-B6, A9-D4 and B9-6, with an antibody potency of 1:1 200 in ELISA; each of the monoclonal antibody ascites prepared had a potency of 1:240,000 after purification;

Analysis 3: Western blot identification of MAbs

DF-1 cells were infected with NDRV ZJOOM, the cells were lysed and the supernatant collected for western blotting with NDRV whole virus as antigen. The results showed that the four MAbs obtained, A5-A1, A5-B6, A9-D4 and B9-6, were all associated with the NDRV

Whole virus responded, with specific bands at approximately 35 kD; they reacted negatively with DF-1-

Cells not infected with NDRV (as shown in Figure 2). The above results show that all four MAbs strains specifically recognize the native SigC protein of NDRV;

Analysis 4: Identification of antigenic epitopes

The synthetic 32 peptides (SigC-1-32) were coated onto ELISA plates and each of the 4 MAbs strains was used as the primary antibody for ELISA detection. The

Results showed that all three monoclonal antibodies, A5-A1, A5-B6 and B9-6, reacted strongly positively with the SigC-18 peptide, weakly positively reacted with the SigC-19 peptide and did not react with the other peptides ( as shown in image 3), and the original antigenic epitopes for these three monoclonal antibodies should be 171SFCSLSPILSGPADA185. The A9-D4 monoclonal antibody did not react with any of the 36 short polypeptides, presumably the antigenic recognized by the A9-D4 monoclonal antibody

Epitope around a conformational spatial epitope. Eight short polypeptides (SigC-18-1-8) synthesized by stepwise shortening of two amino acids at the N- and C-terminus, respectively, were applied to ELISA plates and three MAbs, A5-A1, A5-B6 and B9-6, were used as primary antibodies for ELISA detection, the results showed that the SigC-18 peptide after cleavage of six amino groups at the N-terminal end (corresponding to

SigC-18-5, SigC-18-6 and SigC-18-7 respectively) still showed strong positive reactions with the three monoclonal antibodies A5-A1, A5-B6 and B9-6 (as shown in Figure 4). Therefore, the core antigen epitope corresponding to the three monoclonal antibodies A5-AI, A5-B6 and B9-6 should be 177PILSGPADA185 and consist of 9 amino acids.

Although embodiments of the invention have been shown and described, the

Those skilled in the art that a variety of variations, modifications, substitutions and variants of these

Embodiments are possible without departing from the principles and spirit of the invention, the scope of which is limited by the appended claims and their equivalents.

Claims (10)

Claims LU503352 1. A method for identifying antigenic epitopes of cC protein of novel aquatic reovirus, characterized in that the identification method comprises the following steps: S1: Preparation of raw material Raw materials: cells, virus voice and experimental animals, virus strains including NDRV ZJOOM and CDRV ZJ2000M , cells including chicken embryo filbroblast DF-1; S2: Preparation of Reagents Reagents: pET-28a-his Vector, Test Kit, HRP-labeled Sheep Anti-Mouse IgG (HRP-IgG), Forster's Complete Adjuvant, Forster's Incomplete Adjuvant, PEG4000, Hypoxanthine Thymine Nucleoside , hypoxanthine methotrexate thymine nucleoside; S3: Construction, protein expression and purification of the recombinant plasmid pET-28a-SigC According to the S1 gene sequence of the NDRV ZJ00M strain, a primer pair was designed using the Oligo6.0 software, which uses the SigC ORF of the S1 gene as the target region. and NDRV ZJOOM was taken to inoculate DF-1 cells, 200 µl of cell lysate was taken after 72 hours, RNA extraction was performed, and the target fragment SigC was amplified, and the target fragment SigC was isolated by amplification and pET-28a- His vector to obtain recombinant plasmid pET-28a-SigC; The SigC of the recombinant plasmid pET-28a-SigC and the vector pET-28a-His were subjected to NcoI/XhoI double digestion, the digested products were ligated and transformed, and the resulting recombinant plasmid pET-28a-SigC was double digested and sequencing verified, the validated recombinant plasmid pET-28a-SigC was transformed into receptor cells, and expression was induced by isopropyl-ß-D-thiogalactopyranoside (1.0 mol/L) for 5 h, and centrifuge to collect the bacteria and to perform ultrasonic disturbances, and supernatants and precipitates were separated; Analysis of recombinant protein expression and purification of recombinant proteins, resulting in determination of protein concentration and analysis of its purity by the analyzer; S4: immunization of animals immunization of purified recombinant SigC protein in culture; SS: Cell fusion and preparation of MAbs Mouse spleen cells were fused with mouse myeloma cells by conventional methods, and positive hybridoma cells capable of stably secreting anti-SigC protein-specific MAbss were examined by ELISA, and for the examined positive hybridoma cells stably secreting antibody, ascites was prepared by conventional methods, and the efficacy of the ascites antibody was demonstrated by indirect ELISA; S6: Identification of the biological characteristics of MAbs (1) Identification of MAbs subtypes Identification of the prepared ascites antibodies with the MAbs subtype identification kit; (2) Western blot analysis of MAbs Collection of DF-1 cells infected with NDRV, visualization and analysis of the Western blot bands by kit controls; LUS03352 (3) Indirect Immunofluorescence Identification Conduct control experiments to determine the binding activity of MAbs to NDRV and to assess whether MAbs have binding activity to CDRV and ARV; S7: Identification and localization of antigenic epitopes by peptide scanning A library of 32 SigC-1-32 peptides with a truncated length of 15 amino acids and an overlap of 5 amino acids of the SigC protein was artificially synthesized and each peptide was resolved with DMSO and coated at 1 µg/well, and the ELISA screening was performed with the prepared MAbs as the primary antibody and HRP-labeled sheep anti-mouse IgG as the secondary antibody, and the prepared high immunity anti-SigC protein mouse serum was used as a positive control and the mouse serum without immunity as a negative control for the preliminary identification of the antigenic epitopes by indirect ELISA; The 32 SigC protein peptides (SigC-1-32) were truncated with the peptides that were strong positives in the MAbs ELISA, and the synthesis of 8 SigC-18-1-8 peptides was accelerated by stepwise truncation of the N and C-terminal end continued with a cis shift of 2 amino groups each, and the 8 truncated peptides were coated on ELISA plates, and the antigenic epitope sequence was determined from the OD4s0 value by the same ELISA method as above; S8: Analysis of Conservativity Avian reoviruses such as NDRV and CDRV from different generations and geographic locations were selected and their respective SigC protein amino acid sequences analyzed and compared to examine the conservativity of the antigenic epitopes. 2. A method for identifying antigenic epitopes of the novel aquatic bird reovirus cC protein according to claim 1, characterized in that the kits in step 2 comprise: DL15000 DNA Marker, MiniBEST Viral RNA/DNA Extraction Kit Ver.5.0, PrimeScript™ One Step RT-PCR Kit, Agarose Gel Recovery Kit, Ncol and XhoI Restriction Endonucleases and Plasmid Extraction Kit, MAb Isoform Detection Kit, ECL Chemiluminescence Detection Kit, Ni-NTA Kit. 3. A method for identifying antigenic epitopes of the 6C protein of the novel aquatic reovirus according to claim 2, characterized in that the starting materials in step 1 in particular mouse myeloma cells SP2 / 0, the ARV S1133 strain, E. coli DH5a and BL21 receptor cells and 6-8 week old BALB/c mice. 4. A method for identifying antigenic epitopes of cC protein of novel waterfowl reovirus according to claim 3, characterized in that the target fragment amplifies SigC in step 3 using the extracted RNA as a template with the PrimeScript™ One Step RT-PCR Kit is, PCR reaction system: PrimeScript One Step Enzyme Mix 1 pL, 2xOne Step Buffer 12.5 pL, 20 pmol/L upstream and downstream primers 10 pmol each, RNA template 1.5 pL, supplemented with RNase Free dH20 25.0 uL, where One -Step RT-PCR reaction conditions: reverse transcription at 50°C for 30 min; predenaturation at 94°C for 2 min; Denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 60 s, a total of 30 cycles were performed. 5. A method for identifying antigenic epitopes of the 6C protein of the novel aquatic reovirus according to claim 3, characterized in that in step 3 the expression of the recombinant protein is analyzed by SDS-PAGE, the recombinant protein SUMO- SigC is purified using a Ni-NTA kit, then the protein concentration is determined using a Quawell Q3000 Nucleic Acid Protein Analyzer and its purity is analyzed by SDS-PAGE. 6. A method for identifying antigenic epitopes of the 6C protein of the novel waterfowl reovirus according to claim 3, characterized in that the processing method for the screening by the ELISA method in step 5 comprises: an indirect ELISA Method established in which purified recombinant SigC protein was used as encapsulated antigen, the culture supernatant of the fused hybridoma cells was tested using the established ELISA method, using positive sera from mice with anti-NDRV SigC protein as a positive control and sera from non-immunized Mice were used as a negative control, hybridoma cells corresponding to the ELISA positive supernatant were picked and assayed by the conventional limiting dilution method, and a total of three rounds of subcloning were performed. 7. A method for identifying antigenic epitopes of the 6C protein of the novel waterfowl reovirus according to claim 3, characterized in that the control experiment in step 6 proceeds as follows: After lysis, boiling and centrifugation, the supernatant for the SDS -PAGE electrophoresis, then the PAGE gel is transferred to a nitrocellulose membrane, the NC membrane is sealed with 5% skim milk, and the prepared MAbs as primary antibodies and goat anti-mouse IgG-HRP as secondary antibodies, while DF-1 cells not infected with NDRV were used as a negative control. 8. A method for identifying antigenic epitopes of the 6C protein of the novel aquatic reovirus according to claim 3, characterized in that the immunization culture procedure in step 4 comprises: immunization of 6-8 week old BALB/c mice (100th µg/piece) with purified recombinant SigC protein by multipoint subcutaneous injection in the neck and booster once at an interval of 2 weeks, three times in total, 7 days after the third immunization mice with the highest serum antibody potency (100 µg/piece) were reimmunized with SigC protein (without adjuvant) and spleen cells were fused with SP2/0 cells on the third day. 9. A method for identifying antigenic epitopes of the 6C protein of the novel aquatic reovirus according to claim 7, characterized in that the method of indirect fluorescence identification in step 6 comprises: NDRV ZJOOM was infected with DF-1 cells in an infectious dose of 0.01 MOI and cells not inoculated with the virus were used as a negative control and incubated for 48 hours at 37°C and 5% CO2; the medium was discarded and the cells were fixed with 4% paraformaldehyde and identified by indirect immunofluorescence using the prepared MAbs as the primary antibody and goat anti-mouse IgG-FITC as the secondary antibody to determine the binding activity of the MAbs to NDRV. 10. Fin method for identifying antigenic epitopes of the cC protein of the novel waterfowl reovirus according to claim 9, characterized in that in step 6 DF-1 cells were infected with CDRV ZJ2000M or ARV S1133 at 0.01 MOI and cells that were not inoculated with the virus, used as a negative control, incubated for 48 hours and then identified by IFA to determine whether the MAbs have binding activity for CDRV and ARV, and the western blot bands were analyzed with the ECL chemiluminescence Kit visualized and analyzed.