Antigens, methods, and applications for detecting antibodies against duck reproductive disorder syndrome virus.

Abstract

This invention discloses an antigen for detecting Duck Reproductive Disease Syndrome Virus (DRDV) antibodies, an indirect ELISA detection method based on this antigen, and an ELISA kit for detecting DRDV antibodies. By applying the aforementioned antigen, detection method, or ELISA kit, DRDV antibodies can be effectively identified and their levels detected. As a novel and unknown virus, DRDV allows for efficient and convenient monitoring of DRDV antibody levels through screening for proteins that can serve as its antigens and then applying these proteins to the detection of antibody levels. This provides a convenient and rapid understanding of DRDV infection status in farms, enabling timely prevention and control of DRDV outbreaks, and demonstrates promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology detection, and in particular to an antigen, method, and application for detecting antibodies against duck reproductive disorder virus. Background Technology

[0002] Duck reproductive disorder syndrome (DRS) is a viral disease that is gradually gaining attention in Muscovy duck farming. The pathogen is a novel virus. This virus primarily affects the reproductive system of breeding ducks, leading to decreased egg production, fertilization rate, and hatchability. In severe cases, it can have a lasting negative impact on the reproductive performance and farming efficiency of the breeding flock. Under large-scale Muscovy duck farming conditions, DRS virus can spread among duck flocks through direct or indirect contact. The infection process often lacks obvious specific clinical symptoms, making it easily overlooked in production. Once the virus has spread covertly among breeding ducks, it is often only discovered after a significant decline in egg production, thus increasing the difficulty of prevention and control. However, DRS virus is a novel virus, and its genome sequence and antibody detection targets have not been publicly reported. Therefore, establishing effective monitoring methods for DRS virus infection is a crucial technical aspect of the DRS prevention and control system.

[0003] Serological testing is a commonly used technique in animal viral disease surveillance, offering advantages such as relative simplicity and suitability for large-scale sample testing. Indirect ELISA, due to its high sensitivity, specificity, and reproducibility, has been widely used in poultry disease antibody surveillance. However, the performance of indirect ELISA detection methods largely depends on the selection of the coating antigen. The immunogenicity, specificity, and expression stability of the antigen directly affect the sensitivity and accuracy of the detection method.

[0004] Currently, there is a lack of specific antigens and corresponding ELISA detection methods for detecting duck reproductive syndrome virus (DPRV) antibodies, and a mature, standardized, and practically applicable technical solution has not yet been developed. Therefore, screening DPRV antigens with clear sources and stable immunological characteristics, and establishing a reliable antibody detection method based on these antigens, is of significant practical importance for monitoring DPRV antibody levels in Muscovy duck populations and formulating related prevention and control measures. Summary of the Invention

[0005] The purpose of this invention is to provide an antigen for detecting duck reproductive syndrome virus (DPRV) antibodies, an indirect ELISA detection method for DPRV antibodies, and its application, so as to achieve rapid detection of DPRV antibodies and further realize the monitoring of DPRV antibody levels in Muscovy duck populations and the formulation of related prevention and control measures, which has important practical significance.

[0006] According to a first aspect of the present invention, an antigen for detecting duck reproductive dysgenesis virus (DVRV) antibodies is provided, the antigen being the DVRV ORF4 protein, the amino acid sequence of which is shown in SEQ ID NO:12. Thus, this antigen allows for efficient and specific detection of DVRV antibody levels. As DVRV is a novel and unknown virus, screening for proteins that can serve as its antigens and then applying them to the detection of its antibody levels provides an efficient and convenient way to monitor DVRV antibody levels, enabling quick and easy understanding of DVRV infection status in farms and timely prevention and control of DVRV outbreaks.

[0007] In some embodiments, the duck reproductive disorder syndrome virus is accessed under the number CCTCC NO:V202619.

[0008] In some embodiments, the complete genome sequence of the duck reproductive disorder syndrome virus is shown in SEQ ID NO:1.

[0009] According to a second aspect of the invention, a non-therapeutic application of the antigen in the detection of duck reproductive disorder syndrome virus (DRSV) is provided. This application allows for efficient and convenient monitoring of DRSV antibody levels, providing a quick and easy understanding of DRSV infection status in farms, and enabling timely prevention and control of DRSV outbreaks.

[0010] According to a third aspect of the invention, the use of the antigen in the preparation of products for detecting duck reproductive disorder syndrome virus (DRS virus) is provided. Thus, this application allows for efficient and convenient monitoring of DRS virus antibody levels, providing a quick and easy understanding of DRS virus infection status in farms, and timely prevention and control of DRS virus outbreaks.

[0011] According to a fourth aspect of the present invention, the application of the antigen in the detection of duck reproductive disorder syndrome virus (DRS) antibodies is provided. Thus, this application allows for efficient and convenient monitoring of DRS virus antibody levels, providing a quick and easy understanding of DRS virus infection status in farms, and enabling timely prevention and control of DRS virus outbreaks.

[0012] According to a fifth aspect of the invention, the use of the antigen in the preparation of monoclonal antibodies against duck reproductive disorder syndrome virus (DRS) is provided. This allows for the efficient preparation of DRS monoclonal antibodies.

[0013] According to a sixth aspect of the present invention, an indirect ELISA method for detecting duck reproductive disorder virus antibodies is provided, the method comprising the following steps: S1. Antigen coating: The antigen is coated onto a solid-phase carrier and coated overnight at 4°C. S2. Sealing: Add 5% skim milk powder sealing solution to each well and incubate at 37°C for 2 hours. S3. Add primary antibody: Dilute the serum to be tested at 1:1600 and add 100 μL to each well. At the same time, set up duck reproductive syndrome virus ORF4 positive serum as positive control and duck reproductive syndrome virus ORF4 negative serum as negative control. Incubate at 37℃ for 1 h. S4. Add enzyme-labeled secondary antibody: Add secondary antibody diluted 1:3000 to each well and incubate at 37℃ for 30 min; S5. Color development: Add TMB color development solution to each well and incubate at 37°C in the dark for 10 min. S6. Termination of reaction: Add H2SO4 stop solution to each well to end the reaction; S7. Measurement and Judgment: Read the OD value using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 450 nm: When the OD value of the sample to be tested... 450nm A value greater than or equal to 0.44436 is considered a positive result; when the OD value of the sample is... 450nm When the value is less than 0.44436, the result is considered negative.

[0014] In some embodiments, the antigen coating concentration in step S1 is 0.25 μg / mL.

[0015] Therefore, the indirect ELISA detection method established by this method has good sensitivity, specificity and repeatability. Using it to detect the antibody level of duck reproductive syndrome virus can provide a convenient and quick understanding of the infection status of duck reproductive syndrome virus in farms, and timely prevention and control of duck reproductive syndrome virus outbreaks.

[0016] According to a seventh aspect of the present invention, an indirect ELISA detection method for duck reproductive disorder virus (DPRV) antibodies is provided for non-therapeutic application in the detection of DPRV antibodies. Thus, this application allows for efficient and convenient monitoring of DPRV antibody levels, providing a quick and easy understanding of DPRV infection status in farms, and enabling timely prevention and control of DPRV outbreaks.

[0017] According to an eighth aspect of the present invention, an ELISA kit for detecting antibodies against duck reproductive disorder syndrome virus (DPRV) is provided, the kit comprising the aforementioned antigen. Therefore, this kit allows for efficient and convenient monitoring of DPRV antibody levels, providing a quick and easy understanding of DPRV infection status in farms, and enabling timely prevention and control of DPRV outbreaks.

[0018] According to a ninth aspect of the present invention, a non-therapeutic application of the ELISA kit is provided for monitoring DRDSV antibody levels in Muscovy duck populations. Therefore, this ELISA kit is suitable for monitoring DRDSV antibody levels in Muscovy duck populations and has promising application prospects.

[0019] The beneficial effects of this invention are: 1. This invention discloses an antigen for detecting Duck Reproductive Disease Syndrome Virus (DRDV) antibodies and its application. This antigen has a clear source, high specificity, and can effectively identify DRDV antibodies. DRDV is a novel and unknown virus. By screening for target proteins that can serve as its antigens and then applying them to the detection of antibody levels, DRDV antibody levels can be monitored efficiently and conveniently. This allows for quick and easy understanding of DRDV infection status in farms, enabling timely prevention and control of DRDV outbreaks.

[0020] 2. This invention also discloses an indirect ELISA detection method based on the antigen and its application. This method has good sensitivity, specificity and repeatability. When used to detect the antibody level of duck reproductive disorder syndrome virus, it can conveniently and quickly understand the infection status of duck reproductive disorder syndrome virus in farms and timely prevent and control the outbreak of duck reproductive disorder syndrome virus infection.

[0021] 3. The present invention also discloses an ELISA kit for detecting DRDSV antibodies and its application. The ELISA kit is suitable for monitoring DRDSV antibody levels in Muscovy duck populations and has good application prospects. Attached Figure Description

[0022] Figure 1 Image showing electron microscopic observation of duck reproductive disorder syndrome virus; Figure 2 The results of the alignment of the 7 ORFs of DRDSV with the NCBI virus database sequence; Figure 3The following are the SDS-PAGE results of the expression of seven DRDSV proteins: M is the marker; Lane 1 is the supernatant after centrifugation of ORF1 lysed cells; Lane 2 is the precipitate after centrifugation of ORF1 lysed cells; Lane 3 is the supernatant after centrifugation of ORF2 lysed cells; Lane 4 is the precipitate after centrifugation of ORF2 lysed cells; Lane 5 is the supernatant after centrifugation of ORF3 lysed cells; Lane 6 is the precipitate after centrifugation of ORF3 lysed cells; Lane 7 is the supernatant after centrifugation of ORF4 lysed cells; Lane 8 is the precipitate after centrifugation of ORF4 lysed cells; Lane 9 is the supernatant after centrifugation of ORF5 lysed cells; Lane 10 is the precipitate after centrifugation of ORF5 lysed cells; Lane 11 is the supernatant after centrifugation of ORF6 lysed cells; Lane 12 is the precipitate after centrifugation of ORF6 lysed cells; Lane 13 is the supernatant after centrifugation of ORF7 lysed cells; and Lane 14 is the precipitate after centrifugation of ORF7 lysed cells. Figure 4 Western blotting results for the expression of five DRDSV proteins using His monoclonal antibody: M is the marker, Lane 1 is the supernatant after centrifugation of ORF2 lysed cells, Lane 2 is the supernatant after centrifugation of ORF3 lysed cells, Lane 3 is the supernatant after centrifugation of ORF4 lysed cells, Lane 4 is the supernatant after centrifugation of ORF6 lysed cells, and Lane 5 is the supernatant after centrifugation of ORF7 lysed cells. Figure 5 The Western Blot results for the expression of five DRDSV proteins, incubated with DRDSV-positive serum, are shown in the figure. In the figure, M is the marker, Lane 1 is the supernatant after centrifugation of ORF2 lysed bacterial cells, Lane 2 is the supernatant after centrifugation of ORF3 lysed bacterial cells, Lane 3 is the supernatant after centrifugation of ORF4 lysed bacterial cells, Lane 4 is the supernatant after centrifugation of ORF6 lysed bacterial cells, and Lane 5 is the supernatant after centrifugation of ORF7 lysed bacterial cells. Figure 6 Figure 1 shows the results of DRDSV ORF4 protein expression and purification: M is the marker, Lane 1 is the supernatant after centrifugation of ORF4 lysed cells, and Lanes 2-3 are the protein purification solution. Figure 7 SDS-PAGE results of DRDSV ORF4 protein tag cleavage: where M is the marker, Lane 1 is the protein after enzyme tagged digestion, and Lane 2 is the protein after enzyme tagged digestion and column purification. Figure 8Western Blot results of DRDSV-positive serum incubation before and after DRDSV ORF4 protein tag cleavage: where M is the marker, Lane 1 is the untagged protein, and Lane 2 is the untagged protein. Figure 9 A graph showing the results of determining the critical value for the indirect ELISA method; Figure 10 This is a graph showing the specificity detection results of the indirect ELISA method; Figure 11 This is a graph showing the results of clinical sample testing using the indirect ELISA method. Detailed Implementation

[0023] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various changes and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention.

[0024] Unless otherwise specified, all chemical substances, proteins, enzymes, or reagent kits used in this invention are commercially available.

[0025] Unless otherwise stated, the methods used in this invention, such as PCR amplification and nucleic acid extraction, all employ conventional experimental procedures in the field. Related operations can be performed with reference to commonly used experimental technical manuals or the operating instructions for commercial reagent kits and instruments.

[0026] Example 1: Isolation and whole genome sequence acquisition and analysis of duck reproductive disorder syndrome virus.

[0027] A Muscovy duck farm experienced a decline in egg production. Autopsies of the affected ducks revealed endometrial hemorrhage. Tissue samples from the diseased ducks were collected and tested using existing methods for detecting known pathogens, but no pathogen was detected. The initial diagnosis was an unknown pathogen. Because the main clinical symptoms of this disease are decreased egg production and endometrial hemorrhage, and because the tissue has a strong affinity for the reproductive system, the disease was named "Duck Reproductive Disorder Syndrome." Duck Reproductive Disorder Syndrome, DRDS However, through the isolation of bacteria and mycoplasma from the diseased tissue, no bacteria or mycoplasma could be isolated. Therefore, it was inferred that the cause of the disease was a virus, and the unknown pathogen causing the disease was named "Duck Reproductive Disorder Syndrome Virus (DRS)". Duck Reproductive Disorder Syndrome Virus,DRDSV )".

[0028] DRDSV separation is performed as follows: S1: Separate the oviduct of positive Muscovy ducks and obtain epithelial tissue of the uterine portion of the oviduct: Select 35-week-old positive Muscovy ducks (excluding Muscovy ducks infected with other pathogens and whose clinical symptoms show duck reproductive disorder syndrome). S2: Digestion of Muscovy duck oviduct uterine epithelial tissue: Add 0.25% trypsin + 0.02% EDTA to the Muscovy duck oviduct uterine epithelial tissue from step S1 and let it stand at 37℃ for digestion until the tissue becomes fluffy; S3: Centrifugation to remove blood cells: Add FBS to the digested tissue from step S2 to remove enzymes, centrifuge to discard the FBS, and then add complete culture medium (with a final concentration of 20% FBS, 2-3 mL of penicillin-streptomycin mixture, 2 mmol glutamine, 100 μg / mL heparin sodium, 100 μg epidermal growth factor, 100 μg insulin, and 100 μg IGF-1 recombinant transferrin per 500 mL DMEM / F12 cell culture medium), mix with magnetic beads, filter through a 100 μm cell sieve, centrifuge, discard the supernatant to remove blood cells, and resuspend the precipitated cells; S4: Cell counting and differential adhesion removal of fibroblasts: Count the cells obtained from resuspending in step S3, and seed them in 13.7x10 cm cell culture dishes. 6 The cells were aliquoted and plated, incubated in an incubator for 2 hours, and then the epithelial cells suspended in the cell culture medium were collected and the adherent fibroblasts were removed. S5: Cell plate coating: Coat cell plates with coating solution containing 100% FBS at 37°C; S6: Obtaining primary oviduct epithelial cells: Collect the cell clumps from step S4, suspend them in complete culture medium, and incubate them statically in a 37°C 5% CO2 incubator using the cell plates coated in step S5 to obtain positive Muscovy duck oviduct epithelial primary cells. S7: Collect cell supernatant: Culture the positive Muscovy duck oviduct epithelial primary cells obtained in step S6 and collect the cell supernatant; S8: Isolation of Duck Reproductive Disorder Syndrome Virus: The cell supernatant from step S7 was subjected to sucrose density gradient ultracentrifugation. After centrifugation, the cells were separated into 7 layers. The precipitate layer with 35% sucrose was resuspended and centrifuged again to remove the sucrose. The resuspended precipitate was then used as DRDSV solution, which was then concentrated and purified to obtain DRDSV.

[0029] Electron microscopy was performed on the isolated duck reproductive disorder syndrome virus, and the results are as follows: Figure 1 As shown, electron microscopy revealed viral characteristics, further confirming its status as a virus. The virus was then deposited with accession number CCTCC NO:V202619, under the accession name Duck Reproductive Disorder Syndrome Virus DRDSV-01, and its classification name was... Duck Reproductive Disorder Syndrome Virus The depositary institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, and the deposit date is March 6, 2026. The obtained DRDSV was sequenced, and its whole genome sequence is shown in SEQ ID NO:1.

[0030] Based on the DRDSV whole genome sequence (as shown in SEQ ID NO:1), open reading frame (ORF) prediction analysis was performed on the DRDSV whole genome using bioinformatics software, resulting in 7 predicted ORFs (denoted as ORF1-ORF7). These 7 ORFs were then compared with the GenBank database. Only ORF3 showed a match with the viral rdrp (RNA-dependent RNA polymerase) protein; the other 6 ORFs did not show any match results. Furthermore, the alignment result of ORF3 is as follows: Figure 2 As shown: Figure 2 The first result is a metagenomically assembled genome (MAG), and the remaining results are all rdrp (RNA-dependent RNA polymerase) protein-related sequences of astroviruses, with the highest homology being only 52.13%. This further indicates that the duck reproductive disorder syndrome virus is a novel virus.

[0031] The nucleotide sequences of the seven ORFs (ORF1-ORF7) are shown in SEQ ID NO:2-SEQ ID NO:8, and the amino acid sequences of the seven ORFs (ORF1-ORF7) are shown in SEQ ID NO:9-SEQ ID NO:15.

[0032] Example 2: Construction, expression and purification of DRDSV ORF4 antigen.

[0033] The proteins corresponding to the above 7 ORFs (ORF1-ORF7) were optimized for prokaryotic expression in *E. coli*, and the optimized nucleotide sequences are shown in SEQ ID NO:16-SEQ ID NO:22. Construction of the pSYNO-1-ORF prokaryotic expression plasmid in the prokaryotic expression system: The optimized sequence fragments of the above 7 ORFs (ORF1-ORF7) (shown in SEQ ID NO:16-SEQ ID NO:22) were ligated into the pSYNO-1 backbone vector (NovoPro, V017627), and transformed and purified to obtain the correctly sequenced recombinant plasmids pSYNO-1-ORF1~pSYNO-1-ORF7.

[0034] Prokaryotic expression of recombinant ORF proteins: Plasmids pSYNO-1-ORF1~pSYNO-1-ORF7 with correct sequencing results were transformed into Rosetta bacteria and aseptically plated on LB / kanamycin agar plates and cultured at 37℃ for 18 h. Single clones with positive PCR results were picked and inoculated into 10 mL of LB / kanamycin liquid medium at a 1:100 ratio. The culture was incubated at 37℃ and 200 rpm on a shaker until the OD600nm value reached approximately 0.6. IPTG was then added to a final concentration of 0.4 mmol / L, and expression was induced at 25℃ and 150 rpm for 6 h. 10 mL of the bacterial culture was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL of PBS. Cells were lysed by sonication on ice, centrifuged at 13,000 g for 20 min at 4℃, and the supernatant and precipitate were added to 50 μL of 2× protein loading buffer. The mixture was boiled at 100℃ for 5 min and then analyzed by SDS-PAGE.

[0035] The results are as follows Figure 3 As shown in the figure: the results indicate that soluble expression products of ORF2, ORF3, ORF4, ORF6 and ORF7 proteins can be successfully obtained.

[0036] The five expressed proteins (ORF2, ORF3, ORF4, ORF6, and ORF7) were analyzed by Western blot with His-tagged monoclonal antibodies, and the results are as follows: Figure 4 As shown in the figure, the results indicate that all five proteins can be specifically recognized by the His monoclonal antibody. However, when the five expressed proteins were subjected to Western blot analysis with DRDSV clinically positive duck serum, the results were as follows... Figure 5 As shown, only the ORF4 protein can be specifically recognized by DRDSV-positive serum, indicating that the ORF4 protein has good immunoreactivity. Therefore, the ORF4 protein was selected as the target antigen for the subsequent establishment of an indirect ELISA antibody detection method.

[0037] Large-scale expression and purification of recombinant ORF4 protein: The protein-positive strain was cultured overnight on a shaker and inoculated at a ratio of 1:100 into 300 mL of LB / kanamycin liquid medium. Expression was induced overnight at 20 °C and 150 rpm. After induction, the cells were collected by centrifugation at 4 °C and 4000 g for 10 minutes, resuspended in binding buffer, and lysed by sonication on ice. The cells were then centrifuged at 4 °C and 13,000 g for 20 minutes, and the supernatant was collected. The supernatant was filtered through a 0.42 μm filter and the recombinant ORF4 protein was purified using His-tagged nickel column affinity chromatography. The elution fraction containing the target protein was collected. SDS-PAGE analysis of the lysed supernatant and the purified product showed a clear protein band at approximately 69 kDa, consistent with the theoretical molecular weight (e.g., ...). Figure 6 As shown in the figure, this indicates that the DRDSVORF4 protein was successfully expressed and purified.

[0038] To avoid the influence of the MBP and His tags contained in the pSYNO-1 vector on antibody detection results, the MBP and His tags need to be removed and purified twice: TEV protease is added to remove the N-terminal MBP and His tags. After dialysis overnight, the protein is passed through a Ni-NTA column again to remove the MBP tag, His tag, and uncut protein. SDS-PAGE analysis of the purified product showed a clear protein band at approximately 25 kDa, consistent with the theoretical molecular weight. The purified protein band was uniform, without obvious impurities (such as...). Figure 7 As shown in the figure, this indicates that the DRDSV ORF4 protein purification effect is good. Western blot analysis was performed on the ORF4 protein with MBP and His tags removed and compared with clinically positive DRDSV duck serum. The results are as follows. Figure 8 As shown, the cleaved ORF4 protein was specifically recognized by DRDSV-positive serum, indicating that the cleaved ORF4 protein still possesses good immunoreactivity. The concentration of the cleaved and purified ORF4 protein was determined using a BCA protein quantification kit, showing a concentration of approximately 0.16 mg / mL. The purified protein was aliquoted and stored at −80 °C for later use.

[0039] Example 3: Preparation of DRDSV negative and positive sera: Healthy Muscovy ducks, confirmed negative for both conventional duck pathogen antigens and antibodies, were infected with DRDSV virus to obtain positive serum samples. Blood samples were collected every 7 days post-infection, and the serum was separated and detected using Western blot. Serum samples showing a clear positive reaction in the Western blot were preserved as DRDSV ORF4 positive serum. This was used for the establishment, optimization, and methodological evaluation of the indirect ELISA detection method of this invention.

[0040] Meanwhile, duck serum samples free of common pathogens that have not undergone any immunization or infection treatment were collected. After Western blot analysis confirmed that they did not contain DRDSV ORF4 specific antibody signals, the samples were aliquoted and stored at −80 °C as DRDSV ORF4 negative serum. This was used to establish a negative control system and determine the positive and negative criteria for the detection method.

[0041] Example 4: Establishment and optimization of DRDSV indirect ELISA detection method based on ORF4 antigen.

[0042] 4.1 Indirect ELISA operation steps.

[0043] (1) Antigen coating: Purified DRDSV ORF4 protein was used as the coating antigen. It was diluted with coating buffer (0.05M carbonate buffer) to a certain concentration and added to the microplate at 100 μL per well for coating. After coating, it was washed 3 times with washing buffer (PBST) and patted dry. (2) Blocking: Add 200 μL of blocking solution to each well, incubate at 37℃, discard the liquid, wash 3 times as above, and pat dry; (3) Add primary antibody: Dilute the serum to be tested to a certain concentration and add 100 μL to each well. At the same time, set DRDSV positive serum as positive control and DRDSV negative serum as negative control; incubate at 37℃, wash 3 times as above, and pat dry. (4) Add enzyme-labeled secondary antibody: Add 100 μL of diluted secondary antibody [HRP-labeled Goat Anti-Duck IgG (purchased from KPL Company, USA)] to each well, incubate at 37℃, wash 3 times as above, and pat dry; (5) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37℃ in the dark; (6) Termination of reaction: Add 100 μL of 2M H2SO4 to each well to terminate the reaction; (7) Measurement: The absorbance (OD value) was read at a wavelength of 450 nm using an ELISA reader.

[0044] 4.2 Optimization of ORF4 protein coating concentration and positive / negative serum dilution.

[0045] Purified DRDSV ORF4 protein was used as the antigen and successively diluted with coating buffer to 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, and 0.125 μg / mL. 100 μL was added to each well and the plates were incubated overnight at 4°C. The plates were then washed five times with PBST. Negative and positive control sera were diluted 1:100, 1:200, 1:400, 1:800, 1:1600, and 1:3200 and added to the ELISA plates. Indirect ELISA was performed according to the procedure in section 4.1 to optimize the antigen coating concentration and the dilutions of negative and positive sera. The results are shown in Table 1. When the DRDSV ORF4 protein was coated at a concentration of 0.25 μg / mL and the serum was diluted 1:1600, the P-value was close to 1, and the N-value was relatively small; the P / N ratio of 11.2800 was relatively high. Therefore, the optimized antigen coating concentration was determined to be 0.25 μg / mL, and the optimized serum dilution was determined to be 1:1600.

[0046] Table 1. Optimization of Antigen Coating Concentration and Negative / Positive Serum Dilution

[0047] 4.3 Optimization of ORF4 protein coating conditions.

[0048] The experiments were conducted under the optimized conditions described above. The antigen was coated using three different coating conditions (4°C overnight, 37°C for 1 h, and 37°C for 2 h). Indirect ELISA was performed according to the procedure in 4.1 to optimize the antigen coating conditions. The results are shown in Table 2. The optimized coating condition was overnight coating at 4°C.

[0049] Table 2 Results of ORF4 protein coating condition optimization

[0050] 4.4 Optimization of ORF4 protein blocking conditions.

[0051] The experiment was conducted under the optimized conditions described above, using 5% skim milk powder as the blocking solution. Blocking was performed for 0.5 h, 1 h, and 2 h, respectively, followed by indirect ELISA according to the procedure in 4.1, to optimize the blocking conditions. The results are shown in Table 3. The optimized blocking condition was 5% skim milk powder blocking for 2 h.

[0052] Table 3 Results of ORF4 protein blocking condition optimization

[0053] 4.5 Optimization of primary antibody incubation time.

[0054] The experiment was conducted under the optimized conditions described above. The primary antibody was added to the ELISA plate at the optimized dilution (1:1600), and incubated at 37°C for 0.5 h, 1 h, and 2 h, respectively. Indirect ELISA was performed according to the procedure in 4.1 to optimize the primary antibody incubation time. The results are shown in Table 4. The optimized primary antibody incubation time was 1 h.

[0055] Table 4. Optimization Results of Antibiotic Incubation Time

[0056] 4.6 Optimization of secondary antibody reaction conditions.

[0057] The experiment was conducted under the optimized conditions described above. The enzyme-labeled secondary antibody was diluted 1:2000, 1:3000, and 1:4000, and incubated at 37°C for 15 min, 30 min, and 60 min, respectively. Indirect ELISA was performed according to the procedure in 4.1 to optimize the secondary antibody dilution and reaction time. Table 5 shows that the optimized reaction conditions for the secondary antibody are a 3000-fold dilution, and Table 6 shows that the optimized incubation time is 30 min. In summary, the optimized reaction conditions for the secondary antibody are a 3000-fold dilution at 37°C for 30 min.

[0058] Table 5 Optimization results of secondary antibody reaction concentration

[0059] Table 6 Results of Optimization of Secondary Antibody Incubation Time

[0060] 4.7 Optimization of color development time.

[0061] After determining the coating, blocking, and optimized primary and secondary antibody concentrations and reaction times according to the above steps, 100 μL of commercial TMB substrate solution was added at room temperature in the dark. The reaction times were 5 min, 10 min, and 15 min, respectively. Then, 100 μL of 2M H2SO4 stop solution was added to each well, and the OD450nm value was read. The P / N ratio was calculated, and the development time was optimized. The results are shown in Table 7. The optimized development time was 10 min.

[0062] Table 7 Optimization Results of Color Development Time

[0063] 4.8 Determination of ELISA cutoff values.

[0064] A total of 23 serum samples were confirmed to be negative for both conventional duck pathogen antigens and antibodies. Following the optimized ELISA procedure described in "Example 4", each sample was replicated in triplicate, and the OD values ​​of the negative samples were calculated. 450nm The mean (`X) and variance (SD) of the values ​​are calculated using the formula: Critical value = `X + 3SD. The calculated ELISA OD 450nm The critical value is 0.44436 (e.g.) Figure 9 (As shown). When the OD of the sample to be tested... 450nm A value greater than or equal to 0.44436 is considered a positive result; when the OD value of the sample is... 450nm When the value is less than 0.44436, the result is considered negative.

[0065] In summary, the optimized steps of the DRDSV indirect ELISA detection method based on ORF4 antigen are as follows: (1) Antigen coating: Purified DRDSV ORF4 protein was used as the coating antigen (amino acid sequence as shown in SEQ ID NO:12). It was diluted with coating buffer (0.05M carbonate buffer) to 0.25 μg / mL and added to the microplate, 100 μL per well. The plate was coated overnight at 4°C. After that, it was washed 3 times with washing buffer (PBST) and patted dry. (2) Blocking: Add 200 μL of 5% skim milk powder blocking solution to each well, incubate at 37℃ for 2 h, discard the liquid, wash 5 times as above, and pat dry; (3) Add primary antibody: Dilute the serum to be tested at 1:1600 and add 100 μL to each well. At the same time, set DRDSV ORF4 positive serum as positive control and DRDSV ORF4 negative serum as negative control; incubate at 37℃ for 1 h, wash 5 times as above, and pat dry. (4) Add enzyme-labeled secondary antibody: Add 100 μL of secondary antibody diluted 1:3000 (HRP-labeled Goat Anti-Duck IgG) to each well, incubate at 37℃ for 30 min, wash 5 times as above, and pat dry; (5) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37°C in the dark for 10 min; (6) Termination of reaction: Add 100 μL of 2M H2SO4 to each well to terminate the reaction; (7) Measurement: The absorbance (OD value) of the sample to be tested is read at a wavelength of 450 nm using an ELISA reader: 450nm A value greater than or equal to 0.44436 is considered a positive result; when the OD value of the sample is... 450nm When the value is less than 0.44436, the result is considered negative.

[0066] Example 5: Specificity assay for DRDSV indirect ELISA detection based on ORF4 antigen.

[0067] For the preparation of standard serum, healthy, unvaccinated ducklings aged 9 days were selected for this experiment and divided into an experimental group and a control group. The experimental group was injected intramuscularly with the vaccines described in Table 8 below, and serum was collected two weeks after immunization, which served as the positive standard. At the same time, a non-vaccinated control group was set up, which was raised and its serum was collected under the same conditions, which served as the negative standard.

[0068] Table 8. Vaccine Injection Status of Experimental Group DRV Duck plague virus live vaccine Veterinary Drug Production License No. 180010037 Pulike Biotechnology AIV Avian influenza (H9 subtype) bivalent inactivated vaccine Veterinary Drug Production License No. 221012324 Huapai Biotechnology EDSV Inactivated vaccine for egg drop syndrome (BC14 strain) (2021) Veterinary Drug Certificate No. 71 Intervet / Zorton NDV Newcastle disease inactivated vaccine for chickens (La Sota strain) Veterinary Drug Production License No. 140392146 Jiujiang Bomeile Biotechnology E. coli Chicken E. coli propolis inactivated vaccine Veterinary Drug Production License No. 150101028 Henan Huahong Biotechnology DTMUV Inactivated vaccine for duck Tembusu virus disease (DF2 strain) Ministry of Agriculture and Rural Affairs Announcement No. 473, (2021) New Veterinary Drug Certificate No. 60 Wuhan Keqian Biotechnology PM Inactivated vaccine for avian pasteurellosis (strain 1502) Veterinary Drug Production License No. 151722015 Huabaiwei (Shandong Wohua) Standard positive sera for DRV, AIV, EDSV, NDV, E. coli, DTMUV, and PM, as well as DRDSV positive and negative sera, were collected and detected using the indirect ELISA method established in Example 4. The specificity of the established method was determined by detecting serum cross-reactivity. The detection results are as follows: Figure 10 As shown, the DRDSV ORF4 protein antigen does not react with standard positive serum antibodies against DRV, AIV, EDSV, NDV, E. coli, DTMUV, and PM, indicating that its antigen specificity is good.

[0069] Example 6: Clinical trial of DRDSV indirect ELISA detection based on ORF4 antigen.

[0070] During routine population sampling and monitoring, 10 samples were collected from each of 16 pens of breeding ducks in a certain group to prepare serum for later use. The indirect ELISA procedure established in Example 4 was used for DRDSV antibody monitoring, and the results are as follows: Figure 11 As shown: Antibodies in columns 2, 3, 10, 11, 12, and 13 are significantly elevated.

[0071] Using the optimized conditions (method steps) of the above-mentioned indirect ELISA detection, and using DRDSV ORF4 protein as the coating antigen, to detect DRDSV antibodies, we can quickly and sensitively monitor DRDSV antibody levels, conveniently and quickly understand the infection status of DRDSV in farms, and promptly prevent and control the outbreak of DRDSV infection.

[0072]

[0073] ORF1 nucleotide sequence (SEQ ID NO:2):

[0074] ORF2 nucleotide sequence (SEQ ID NO:3):

[0075] ORF3 nucleotide sequence (SEQ ID NO:4):

[0076] Nucleotide sequence of ORF4 (SEQ ID NO:5): ATGAATCCAAGTGCTCCAGTGTGGACACCGCAGAATAGTAATGGTAATAGTGTTGATGGTCCTCCGTATGGTTATACTAATCCACCGTTTCAGGCTTACCCTTGGGGTAATTTACCATCTAATCGGAATTGGTATCCCAGAAGAGGATGGTGTAGACCATGGCGTGGTCGTGGCAACTGGAATAATAGGAATCAAGACCGCGGGAGATCTCGTGATCGCGCATCATCAAGAGCTCGTTCGAGGAGTCGATCTGCTAGTCGTCGGCGTGGACATGGAGACGACTGGTACAGCCAGTGGGGACACAATGTCGTAACGTATGGTATGGTAACTGTGACACAGCAAGGGAAGTCTAATTTTAATCTTAGGAATCCCTGGAATTATAATAATAAGGACGTTGAACAGACAAAGTTGAGAGACTGGAGATTTCCAGCATCCACTGAAGTTGAAACAGTTGTCTTTGATGGTGATAACAATGGTGGTTTCTATCGTGTTGGGTTGAAAGTTGATATTGCTTCTCCTGATTCGAGGAAAGTAAGATTTGGTACACCTACTGATTTGGCAGATGGTGTTAAAAATTTAACTCTTGATTCTGAAGGCGATAAGAAGAAAAAGCGTGGTGGTATCTTTAGGAGATGGCGTTCCACGGAGCAGTTGTGA。

[0077] Nucleotide sequence of ORF5 (SEQ ID NO:6): ATGATGATCACCATCAATGCACACAACACACTTCGTTTGTTCCTCACAACACAACACCTTACAACCCATACGACCACGATTGCGATCAACAAGTGTTTTGCCAAACCAATCATCGATTTTATTATTACACTTCTTACAACATCTCTCTTTTGCATATTTACAGTCACGAACACTGTGACCAACAACGTTACACCAAATACAGGCTTGGCCAATACAAACATCACAATCACAATTCTTATCACACTGACACCTTGGACAATGACAATCACCATTCAAATTCGAATGTTTACTAATGCCACCACCAACAAACAATTGTTTTTTATCAAATTCACTACTAGATGGCTTAACCTTAGACTCCTGCCTTCTCCGCTGAGGCAAGGCTTCAACAACATCCTGCCTCCTCCGCTGTGGCAAAACTTCAACAACATTTTCCTCAACCGGTAA。

[0078] ORF6 nucleotide sequence (SEQ ID NO:7): ATGCATAACAGCATTATCCAATTCCAAAATGCTCCATCTCTCTTGACCTTAGCAACAAGAGGTCCACGAAAAATTTTAACAACCTTACCATCACAAAGACATGACAACAACCTCTCTCTAACCTCAACAAAACACAACAAAGCCGGATTAATTACTACCACCACCCACGACACAGGTGTTGCAATCCACGGAACAAAAAGCAAAACCAAAATCATAACACAAAACCAAATTCCATTAATAATACCAACATCCATAACACACCAAAACACAAAGACACCAACAAGTGATTTCACATCTCTCATGCCTCGCATATGA。

[0079] ORF7 nucleotide sequence (SEQ ID NO:8): ATGACAAGCCATGATGATCACCATCAATGCACACAACACACTTCGTTTGTTCCTCACAACACAACACCTTACAACCCATACGACCACGATTGCGATCAACAAGTGTTTTGCCAAACCAATCATCGATTTTATTATTACACTTCTTACAACATCTCTCTTTTGCATATTTACAGTCACGAACACTGTGACCAACAACGTTACACCAAATACAGGCTTGGCCAATACAAACATCACAATCACAATTCTTATCACACTGACACCTTGGACAATGACAATCACCATTCAAATTCGAATGTTTACTAA。

[0080] ORF1 amino acid sequence (SEQ ID NO:9):

[0081] Amino acid sequence of ORF2 (SEQ ID NO:10): MAFHGAVVICLGFLNLCYCDPFLNFGFSLGWFNDTKVNGSNDSNVTTTSLSTAFGSIQGSVWGVVFGNQDSSGVVYVDWAGRTGGFVGADQIFNFTGNHSALGCNWSSNDTLVAGGPRPPGTRVPQFDNVTVDCGDLAVRLDFGGGSKLICGIDSKEDRHRGQVIGGITFGCDDSGCSSTSYYFRCYGNRSMEIDSNCTNGGGNNTNGTRCGKPVVKRWSFVRPVEEVSDKWNFTDKIVVCSYDCWGFSCADFNNCTCTGGPGAICCSTGNNMTSCRVLGDGTETWWSHHFEVIGLGLIVFLILVICVEIVLFCCGFKRKKKRRWLITNGETTIEAKGWFFVFLLWYCGFRSATAISIGKNDLNRWFSGPEDFGFKVTSLGFVANGSKFEERCCQWGHSTAGGISCAVDTPDNDNDPCYKWSQDPTQWDCRSYENGVAGWNDGCFLENGNTVSWCCKCRCRSSWTGWRFKFTGKPVGSIMLRSANHTLVVNISGDGTYYTAFANVTVSGCTGPMALDAWVSRGHWLDAHSCGLSIGDRPLHGAVHCSGRSNWGSVVTSCYCPQGGELGPISVDVDLGFPYKQKNVPAARVSGTCDVNFEWVEGKQNLGNCSLWCGDWSDGWCQVHIMNDTCIFQNVTQTKGITWIYCGNDMIIGKSKCNGTVWRPTVPGPYNPDEKHDSEHDNGFAHLWKEFLEFWQKWFYWIVIVIIVFVFWLIVRLLFCCNRK。

[0082] Amino acid sequence of ORF3 (SEQ ID NO:11): MIFEWDKDFVKNFYKYLEDKMVKCCGMVNNFPHSIFNRDVVHGLRYLIDNDCNERVVDEFKPGNGFEWLSTGKYVRESGYVYGEYTTRDYIEDMFLKRFYESPFYPELRFNKLSKDFGVKYCRGLISVGMMPSRIRDVCKVNYPFEAEVPSVDLEAERFFKNYDWCNKIIGAYSWPKICLKSEMESVARFCPDERLCSGLEGLKFNSILIAFNYCVEDWNMGRSSVGLSDEDFVVTNRSWTNSPGYPYNKNFCRQAREAFDVYFGLLKHYEYSSMNDWMPTVFNVFCKKEILKAEKVLANDIRTIIAPALCQQLVMQKLTLAISMRVSSNWRFSHTSIGRTRWKDDVDFTGCRIGRFDFICEYDISKWDRSIKSFLLKLFWFYCWFVIDTTKLEHFWQLSNGFESVIYSFMLHKNGEVIRKHYGVPSGFTLTSYVNSWVHTFLNVLCFWELCPVECDINYFKKHCDFVCYGDDGLMGISKEAAEWFTIDARSEWLKTKWGITMDPKKCKMVDRYYFTFDGVDVDGIMFLGDVMKVDEDMGRIVPVFKISKIINQFILGNAVKNYMPSEKILICFGHYVECFFHPNCEVIREYLCFLMNKYKYSYIMKTMFKSEYVEYLAMTNNDLICKIRSLCFDRDKFRAFIMNMFYSVKDESKCSSVDTAE。

[0083] Amino acid sequence of ORF4 (SEQ ID NO:12): MNPSAPVWTPQNSNGNSVDGPPYGYTNPPFQAYPWGNLPSNRNWYPRRGWCRPWRGRGNWNNRNQDRGRSRDRASSRARSRSRSASRRRGHGDDWYSQWGHNVVTYGMVTVTQQGKSNFNLRNPWNYNNKDVEQTKLRDWRFPASTEVETVVFDGDNNGGFYRVGLKVDIASPDSRKVRFGTPTDLADGVKNLTLDSEGDKKKKRGGIFRRWRSTEQL。

[0084] Amino acid sequence of ORF5 (SEQ ID NO:13): MMITINAHNTLRRLFLTTQHLTTHTTTIAINKCFAKPIIDFIITLLTTSLFCIFTVTNTVTNNVTPNTGLANTNITITILITLTPWTMTITIQIRMFTNATTNKQLFFIKFTTRWLNLRLLPSPLRQGFNNILPPPLWQNFNNIFLNR.

[0085] ORF6 amino acid sequence (SEQ ID NO:14): MHNSIIQFQNAPSLLTLATRGPRKILTTLPSQRHDNNLSLTSTKHNKAGLITTTTHDTGVAIHGTKSKTKIITQNQIPLIIPTSITHQNTKTPTSDFTSLMPRI.

[0086] ORF7 amino acid sequence (SEQ ID NO:15): MTSHDDHHQCTQHTSFVPHNTTPYNPYDHDCDQQVFCQTNHRFYYYTSYNISLLHIYSHEHCDQQRYTKYRLGQYKHHNHNSYHTDTLDNDNHHSNSNVY.

[0087] ORF1 codon-optimized nucleotide sequence (SEQ ID NO:16):

[0088] nucleotide sequence after codon optimization for ORF2 (SEQ ID NO:17):

[0089] nucleotide sequence optimized for ORF3 codon (SEQ ID NO:18):

[0090] Nucleotide sequence of optimized ORF4 codons (SEQ ID NO:19): ATGAACCCGAGCGCGCCGGTTTGGACCCCGCAGAACAGCAACGGCAACTCTGTTGATGGCCCGCCGTACGGCTACACCAACCCGCCGTTCCAGGCGTACCCGTGGGGCAACCTGCCGTCTAACCGTAACTGGTACCCGCGTCGTGGCTGGTGCCGTCCGTGGCGTGGCCGTGGCAACTGGAACAACCGTAACCAGGATCGTGGCCGTAGCCGTGATCGTGCGAGCAGCCGTGCGCGCAGCCGTTCTCGCAGCGCGAGCCGTCGTCGTGGCCACGGCGATGATTGGTACAGCCAGTGGGGCCACAACGTTGTTACCTACGGTATGGTTACCGTTACCCAGCAGGGCAAAAGCAACTTCAACCTGCGTAACCCGTGGAACTACAACAACAAAGACGTGGAACAGACCAAACTGCGCGACTGGCGCTTCCCGGCGTCCACCGAAGTGGAAACCGTGGTGTTCGATGGCGACAACAACGGCGGCTTCTACCGTGTCGGTCTGAAAGTTGACATCGCTTCTCCGGATAGCCGGAAAGTGCGTTTTGGTACGCCGACCGATCTGGCGGACGGCGTGAAAAACCTGACCCTGGATTCTGAAGGTGATAAGAAGAAAAAACGCGGTGGTATTTTCCGTCGTTGGCGTTCCACCGAGCAGCTGTAA。

[0091] Nucleotide sequence of optimized ORF5 codons (SEQ ID NO:2*): It should be noted that there seems to be a typo in the original text where "SEQ ID NO:2*" should probably be "SEQ ID NO:20".ATGATGATCACCATCAACGCTCACAACACCCTGCGTCTGTTCCTGACCACCCAGCATCTGACCACTCACACCACCACCATTGCTATTAACAAATGTTTCGCTAAACCGATTATTGATTTTATTATTACCCTGCTGACTACCTCTCTGTTCTGCATTTTCACTGTTACCAACACCGTTACCAACAACGTTACCCCGAACACCGGTCTGGCGAACACCAACATTACCATTACCATCCTGATTACCCTGACTCCGTGGACCATGACCATTACCATTCAGATTCGTATGTTTACCAACGCTACCACCAACAAACAGCTGTTCTTCATTAAATTCACCACCCGTTGGCTGAACCTGCGTCTGCTGCCGTCTCCGCTGCGTCAGGGTTTTAACAACATCCTGCCGCCGCCGCTGTGGCAGAACTTCAACAACATTTTCCTGAACCGTTAA。

[0092] [[ID= 3]]Nucleotide sequence after codon optimization of ORF6 (SEQ ID NO:21): ATGCACAACAGCATCATCCAGTTTCAGAACGCGCCGAGCCTGCTGACCCTGGCGACCCGTGGCCCGCGTAAAATCCTGACCACCCTGCCGAGCCAGCGACACGATAACAACCTGAGCCTGACCAGCACCAAACACAACAAAGCAGGCCTGATCACCACCACCACCCACGATACCGGAGTTGCGATCCACGGCACCAAAAGCAAAACCAAAATCATCACCCAGAACCAGATCCCGCTGATCATCCCGACCAGCATCACCCACCAGAACACCAAAACCCCGACCAGCGACTTCACCAGCCTGATGCCGCGCATTTAA。

[0093] Nucleotide sequence after codon optimization of ORF7 (SEQ ID NO:22): ATGACCAGCCACGATGACCACCACCAGTGCACCCAGCACACCAGCTTCGTTCCGCACAATACCACCCCGTACAACCCGTACGACCACGACTGCGATCAGCAGGTTTTCTGCCAGACCAACCATCGCTTCTACTACTATACCAGCTACAACATCAGCCTGCTGCACATCTACAGCCACGAACACTGCGACCAGCAACGTTACACCAAATACCGTCTGGGTCAGTACAAACATCACAATCACAACAGCTATCACACCGATACCCTGGATAACGACAACCACCACAGCAACTCCAACGTTTATTAA。

Claims

1. Antigens for detecting duck reproductive disorder syndrome virus antibodies, among which, The antigen is the ORF4 protein of duck reproductive disorder syndrome virus, and the amino acid sequence of the protein is shown in SEQ ID NO:

12.

2. The antigen according to claim 1, wherein, The accession number of the duck reproductive disorder syndrome virus is CCTCC NO:V202619.

3. The antigen according to claim 1, wherein, The complete genome sequence of the duck reproductive disorder syndrome virus is shown in SEQ ID NO:

1.

4. The use of the antigen according to any one of claims 1-3 in the preparation of a product for detecting duck reproductive disorder virus.

5. The use of the antigen according to any one of claims 1-3 in the preparation of monoclonal antibodies against duck reproductive disorder virus.

6. An ELISA kit for detecting antibodies against duck reproductive disorder virus, wherein, The kit includes the antigen as described in any one of claims 1-3.

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