Monoclonal antibody of duck reproductive disorder syndrome virus, preparation method and application

Abstract

The application discloses a duck reproductive disorder syndrome virus monoclonal antibody. The monoclonal antibody can be used as a key tool for analyzing the new virus structure and function of the duck reproductive disorder syndrome, and is the basis for developing high-sensitivity and high-specificity ELISA, colloidal gold and other diagnostic methods. Meanwhile, the monoclonal antibody is a core reagent raw material for detecting and diagnosing the new virus, and can be used for virus typing, variation strain identification and animal tracing, and is a core tool for disease control and pathogen monitoring. The monoclonal antibody can realize early, rapid and high-throughput detection, support epidemiological research, promote source innovation of vaccine target and design, and can be directly applied to clinical treatment. Moreover, the monoclonal antibody can also be used for preparing a medicine for preventing and treating the duck reproductive disorder syndrome virus, and is used for preventing and treating the virus.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody preparation technology, and in particular to a monoclonal antibody against duck reproductive disorder virus, its preparation method, and its application. Background Technology

[0002] Recently, Muscovy ducks have experienced an unexplained and abnormal drop in egg production. Research has led to the discovery of an unknown duck reproductive disorder syndrome virus (DPRS virus), which has caused significant economic losses to waterfowl farming. Monoclonal antibodies play a crucial role in the detection of emerging diseases. Monoclonal antibodies that bind to the natural structure of viruses are key tools for elucidating the structure and function of new viruses. They are also essential for developing highly sensitive and specific diagnostic methods such as ELISA, colloidal gold, chemiluminescence, and immunofluorescence. Furthermore, they serve as core reagents for the detection and diagnosis of emerging viruses, used for virus typing, variant identification, and animal tracing. They are core tools for disease control and pathogen surveillance, enabling early, rapid, and high-throughput detection, supporting epidemiological research, promoting original innovation in vaccine targets and design, and even being directly applied to clinical treatment. Currently, there are few research reports related to DPRS virus. This invention discloses a monoclonal antibody against DPRS virus (an unknown virus), its preparation method, and its applications, which can lay the foundation for the prevention of this disease. Summary of the Invention

[0003] The purpose of this invention is to provide a monoclonal antibody against duck reproductive disorder syndrome virus, its preparation method, and its application, laying the foundation for the detection, diagnosis, and prevention of this new virus.

[0004] According to a first aspect of the present invention, a monoclonal antibody against duck reproductive disorder syndrome virus (DRS) is provided, wherein the heavy chain variable region amino acid sequence of the monoclonal antibody is MGWSCIILFLVATGTGVHSQVQLQQPGAELVRPGASVKLSCKASVYTFTSYWMSWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCDRYDDTVDYWGQGTSVTVSS, and the light chain variable region amino acid sequence is MMSPAQFLFLLVLWIRETNGDVVLTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTNLEIK. Therefore, this monoclonal antibody can achieve efficient and specific detection of DRS and can also be used for the prevention and control of DRS.

[0005] In some embodiments, the duck reproductive syndrome virus has the accession number CCTCC NO:V202619, and the monoclonal antibody can specifically bind to the duck reproductive syndrome virus.

[0006] According to a second aspect of the present invention, a nucleotide sequence encoding the aforementioned monoclonal antibody is provided. Thus, this nucleotide sequence allows for efficient in vitro expression of the monoclonal antibody after transfection with a vector, enabling mass production and enhancing its application value.

[0007] According to a third aspect of the present invention, the application of the above-described monoclonal antibody in the preparation of a detection kit for duck reproductive disorder syndrome virus (DRS). Thus, the kit allows for efficient and specific detection of DRS.

[0008] According to a fourth aspect of the present invention, the use of the above-described monoclonal antibody in the preparation of a drug for the prevention and treatment of duck reproductive disorder syndrome virus (DRS). Thus, through this application, a drug for the prevention and treatment of DRS can be prepared for the prevention and treatment of the virus.

[0009] According to a fifth aspect of the present invention, a kit is provided containing the aforementioned monoclonal antibody. Thus, this kit enables efficient and specific detection of duck reproductive disorder syndrome virus.

[0010] According to a sixth aspect of the invention, a medicament is provided containing the aforementioned monoclonal antibody. Thus, the medicament can be used for the prevention and treatment of the virus.

[0011] According to a seventh aspect of the present invention, a method for preparing a monoclonal antibody against duck reproductive disorder virus is provided, the method comprising the following steps: S1: The duck reproductive disorder syndrome virus was isolated and then sequenced to obtain the whole genome sequence; S2: Analyze the whole genome sequence obtained in step S1 and screen out protein sequences that can be used as antigens. The protein is ORF4 protein, and its amino acid sequence is shown in SEQ ID NO:12. S3: Immune mice with the antigen protein selected in step S3; S4: Perform serum testing on immunized mice to screen for positive immunized mice; S5: Obtain B cells from positive immunized mice and screen for antigen-affinity single B cells by flow cytometry; S6: Amplify the paired variable region sequences of light and heavy chains from the obtained antigen-affinity single B cells and construct paired light and heavy chain plasmids; S7: Transfect cells with paired light and heavy chain plasmids, and then screen to obtain clones with the highest positive titers; S8: The paired light and heavy chain plasmids corresponding to the clones obtained in step S7 are transfected into CHO cells for in vitro expression and purification to obtain in vitro prepared monoclonal antibodies. Therefore, this method allows for the efficient and specific preparation of monoclonal antibodies against duck reproductive disorder syndrome virus, which can then be used for the detection, diagnosis, and prevention of this virus.

[0012] In some embodiments, the complete genome sequence of the virus in step S1 is shown in SEQ ID NO:1.

[0013] According to an eighth aspect of the present invention, a monoclonal antibody prepared by the above-described method is provided, which specifically binds to duck reproductive disorder syndrome virus. Therefore, this monoclonal antibody can be used for the detection, diagnosis, and prevention of the virus.

[0014] According to a ninth aspect of the present invention, the use of the above-described monoclonal antibody in the preparation of a duck reproductive syndrome virus detection kit or in the preparation of a drug for the prevention and treatment of duck reproductive syndrome virus is provided.

[0015] The beneficial effects of this invention are as follows: This invention provides a monoclonal antibody against duck reproductive syndrome virus (DRSV), its preparation method, and its applications. This monoclonal antibody can serve as a key tool for analyzing the structure and function of this novel DRSV; it is also the foundation for developing highly sensitive and specific diagnostic methods such as ELISA, colloidal gold, chemiluminescence, and immunofluorescence; simultaneously, it is a core reagent raw material for the detection and diagnosis of this emerging virus; it can be used for virus typing, variant identification, and animal tracing, making it a core tool for disease control and pathogen monitoring; it enables early, rapid, and high-throughput detection, supports epidemiological research, promotes original innovation in vaccine targets and design, and can even be directly applied to clinical treatment. This monoclonal antibody specifically binds to the virus's ORF4 protein with strong binding ability and can also bind to natural viruses, making it applicable to various laboratory scenarios such as ELISA, Western blot, and IFA, showing promising prospects. Moreover, this monoclonal antibody can also be used to prepare drugs for the prevention and treatment of DRSV, reducing the incidence of the virus in Muscovy ducks and thus improving their production performance. Attached Figure Description

[0016] 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: 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 The following is a Western blotting result of incubation with His monoclonal antibody to express five DRDSV proteins: 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. 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 6 Figure 1 shows the results of DRDSV ORF4 protein expression and purification: Lane 1 is the supernatant after centrifugation of lysed ORF4 cells, and Lanes 2-3 are the protein purification solutions. Figure 7 SDS-PAGE results of DRDSV ORF4 protein tag cleavage: Lane 1 is the protein after tag cleavage, and Lane 2 is the protein after tag cleavage and column purification. Figure 8 Western Blot results of DRDSV-positive serum incubation before and after DRDSV ORF4 protein tag cleavage: Lane 1 is the untagged protein, and Lane 2 is the untagged protein. Figure 9The image shows the SDS-PAGE results of ORF4 monoclonal antibody expression: Lane M is the marker, Lane 1 is the supernatant after centrifugation, Lane 2 is the supernatant after centrifugation of blank cells; Lane 3 and Lane 4 are 0.1M glycine elution buffer. Figure 10 The results of Western blot analysis of the ORF4 monoclonal antibody; Figure 11 The results show the indirect immunofluorescence assays of ORF4 monoclonal antibody and ORF4 protein; the left image shows the positive control results, and the right image shows the negative control results. Figure 12 The results show the indirect immunofluorescence detection of ORF4 monoclonal antibody and duck reproductive disorder syndrome virus; the left figure is the positive control result, and the right figure is the negative control result. Detailed Implementation

[0017] 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.

[0018] Unless otherwise specified, all chemical substances, proteins, enzymes, or reagent kits used in this invention are commercially available.

[0019] 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.

[0020] Example 1: Isolation and whole genome sequence acquisition and analysis of duck reproductive disorder syndrome virus.

[0021] 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 were found. 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).

[0022] DRDSV separation is performed using the following method: 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, then centrifuge to discard the FBS. Add complete culture medium (20% FBS, 2-3 mL 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.

[0023] Electron microscopy was performed on the isolated duck reproductive disorder syndrome virus, and the results are as follows: Figure 1As 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 in 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.

[0024] 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.

[0025] 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.

[0026] Example 2: Construction, expression and purification of DRDSV ORF4 antigen.

[0027] 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.

[0028] Prokaryotic expression of recombinant ORF protein: 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 incubated at 37℃ for 18 h. Single clones with positive PCR results were picked and inoculated at a 1:100 ratio into 10 mL of LB / kanamycin liquid medium and cultured on a shaker at 37℃ and 200 rpm until OD500 was reached. 600nm When the value was approximately 0.6, IPTG was added to a final concentration of 0.4 mmol / L, and expression was induced at 25℃ and 150 r / min for 6 h. 10 mL of bacterial culture was centrifuged at 8000 r / min for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL 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, boiled at 100℃ for 5 min, and then analyzed by SDS-PAGE.

[0029] 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.

[0030] 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.

[0031] Large-scale expression and purification of recombinant ORF4 protein: The protein-positive strain was cultured overnight on a shaker and then 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.

[0032] 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 ORF4 protein was aliquoted and stored at −80 °C for later use.

[0033] Example 3: Obtaining ORF4 protein antigen-specific memory B cells.

[0034] 3.1 Immunization and titer determination in mice.

[0035] Balb / c mice were subcutaneously immunized. ORF4 protein (antigen) was injected at four subcutaneous points on the back and two subcutaneous points in the groin. The injection dose was 25 μg / 100 μL per mouse. The second immunization was performed 14 days after the first immunization, using the same method as the first immunization. The third immunization was performed one week after the second immunization, using the same method as the first immunization. Two days after the third immunization, blood was collected from the tail vein of the mice to determine the antibody titer. The determination method is as follows.

[0036] (1) ORF4 protein coating: The antigen (ORF4 protein) was diluted to 2 μg / mL and added to 100 μL per well of a 96-well microplate. The plate was incubated overnight at 4°C. The coated microplate was washed three times with a plate washer and drained. Blocking buffer was added to 100 μL per well and incubated at 37°C for 2 hours. The blocked microplate was then washed three times with a plate washer and patted dry.

[0037] (2) Incubation of primary antibody: Dilute the immunized mouse serum with PBS buffer. The pre-immunization mouse serum is the negative control, and the triple-immunization serum is the positive control. The dilution ratios of mouse serum are 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, and 1:512000. Add 100 μL of the diluted serum to the corresponding wells and incubate at 37°C for 1 h. After incubation, wash the microplate three times in a plate washer and pat dry.

[0038] (3) Incubation of secondary antibody: Add 100 μL of HRP-labeled goat anti-mouse secondary antibody to each well and incubate at 37°C for 30 min. Then, wash the plate three times in a plate washer and dry it.

[0039] (4) Color development: Add 100 μL of the mixed color development solution to each well of the 96-well plate and incubate at 37°C for 10 min.

[0040] (5) Termination and reading: Add 100 μL of stop solution to each well of the 96-well plate, and then place it in a microplate reader for reading. The detection wavelength is set to OD. 450nm Read the test results; an OD value greater than 2.1 times that of the negative control is considered positive. A titer of 512,000 meets the sorting criteria and proceeds to the next test. If the titer does not reach 512,000, a booster immunization via peritoneum is administered 7 days after the third immunization.

[0041] Intraperitoneal booster immunization: Intraperitoneal injection, with an insertion angle of 30 degrees and a depth of 5 mm. After confirming that the needle tip has not pierced the peritoneal contents, the ORF4 protein antigen is slowly injected. The injection dose is 25 μg / 100 μL per mouse. Two days after immunization, blood is collected from the tail vein of the mice for antibody titer determination. The determination method and judgment criteria are the same as described above.

[0042] 3.2 Flow cytometry shock sorting of antigen-specific memory B cells.

[0043] (1) Collect the spleens of immune mice that are positive for serum and have a titer of 512,000.

[0044] (2) Preparation of B cell suspension: Transfer the spleen to the cell filter and gently press the spleen with the rubber end of the syringe plunger to obtain cell suspension; place the centrifuge tube in a centrifuge at 1500 rpm for 10 minutes at room temperature; discard the supernatant, add 3 mL of red blood cell lysis buffer and incubate on ice for 12 minutes; add SOL001 to 45 mL and centrifuge at 1500 rpm for 10 minutes at room temperature; discard the supernatant and repeat the washing once; discard the supernatant, resuspend the cells and store at 4℃ for later sorting.

[0045] (3) Fluorescent labeling: 1) Add Live / Dead dye (dilute according to the instructions, usually 1:1000), incubate at room temperature in the dark for 15 min, add staining buffer to stop, centrifuge and discard the supernatant.

[0046] 2) FITC-antigen staining (for identifying antigen-affinity B cells): Replace the ORF4 protein buffer with 0.1 M NaHCO3–Na2CO3 buffer (pH 8–9), add NHS-FITC reagent (FITC active ester), and incubate at room temperature in the dark for 30 minutes. Remove unreacted free FITC by ultrafiltration, and resuspend the cells in staining buffer. Add FITC-ORF4, 0.5 μg / mL, and incubate at 4°C in the dark for 30 minutes.

[0047] 3) PE-CD19 staining (recognition of B cells): Add PE-CD19 antibody directly to the above system, dilute 1:100 and incubate at 4°C in the dark for 20 min.

[0048] 4) Wash to remove free fluorescence. Add 2 mL of staining buffer to a centrifuge at 1500 rpm for 10 minutes at room temperature. Discard the supernatant and repeat the washing process twice. Finally, resuspend the solution in staining buffer and filter through a 40 μm filter for later use.

[0049] (4) Sorting: On a flow cytometer, single live B cells that are antigen-affinity are sorted into 96-well plates by detecting the fluorescence signal of the antigen protein and the fluorescence signal after B cell staining.

[0050] Example 4: Obtaining the variable region sequence of ORF4 monoclonal antibody.

[0051] 4.1 Obtaining the variable region sequence of light and heavy chains in pairs.

[0052] (1) Reverse transcription: B cells were lysed to release RNA. After standing at room temperature for 5–10 min, cDNA was obtained by reverse transcription. 8 μL of single-cell lysis buffer was added to each well of the 96-well plate. After sorting, the cells were briefly centrifuged at 1000 rpm for 10 s and stood at room temperature for 10 min. 2 μL of 5X PrimeScript RT Master Mix (Perfect Real Time) was added according to the instructions. The reverse transcription system was prepared by gently mixing and then performing the reverse transcription reaction. The PCR instrument was set to the following conditions: 37℃ for 15 min (reverse transcription reaction), 85℃ for 5 sec (reverse transcriptase inactivation reaction), and stored at 4℃ for later use.

[0053] (2) Obtaining the variable region sequences of the light and heavy chains: The PCR instrument was programmed, and the variable region sequences were obtained using PCR-specific primers for the light and heavy chains with added restriction enzyme sites. PCR amplification was performed according to the instructions of the Thermo SuperScript™ IV Single Cell / Low-Input cDNAPreAmp Kit (catalog number: 11754050). The RNA from the sorted single B cells was reverse transcribed into cDNA, and the IgG antibody heavy chain and light chain variable region genes were amplified by two rounds of nested PCR. The amplification primer sequences are shown in Tables 1 and 2. The primers used for the first round of heavy chain PCR amplification were IgH-OF1 ~ IgH-OF11 and IgH-OR1, and the primers used for light chain PCR amplification were Igκ-OF1 ~ Igκ-OF10 and Igκ-OR1. The second round of PCR amplification used the products from the first round of PCR amplification as templates, and amplification was performed using primers IgH-IF, IgHIR and Igκ-IF, Igκ-IR (sequences shown in Tables 1 and 2). PCR amplification was performed according to the Thermo Phusion™ Plus Green PCR Master Mix (catalog number: F659) instructions. The reaction system consisted of: 10 µL of Phusion™ Plus Green PCR Master Mix, 1 µL each of the forward and reverse primers, 1.5 µL of template, and nucleic acid-free water to a total volume of 25 µL. The PCR amplification program was: 98 ℃ for 30 s; 98 ℃ for 10 s, 60 ℃ for 10 s, 72 ℃ for 30 s, 35 cycles; 72 ℃ for 5 min.

[0054] Table 1 Primers for PCR amplification of the variable region gene of mouse IgG antibody heavy chain

[0055] Note: The primer sequence contains degenerate bases, where S = C or G, Y = C or T, R = A or G, and W = A or T.

[0056] Table 2 Primers for PCR amplification of mouse IgG antibody light chain variable region gene

[0057] Note: The primer sequence contains degenerate bases, where S = C or G, Y = C or T, R = A or G, and W = A or T.

[0058] (3) Construction of light and heavy chain paired candidate antibody plasmids: ligation and transformation to obtain monoclonal bacteria, and colony PCR screening of light and heavy chain paired candidate antibody clones.

[0059] Prepare a 1% agarose gel, and respectively load the above PCR products into the gel loading wells; complete the recovery and purification of the target fragment according to the instruction manual of the E.Z.N.A.® Gel Extraction Kit (brand: Omega Bio-Tek, catalog number D2500-01).

[0060] The pcDNA3.1 vector is sourced from Sangon Ditech Biotechnology Co., Ltd. Use BamHⅠ (BamH I (conventional type), brand: NEB, catalog number: R0136) and XhoⅠ (Xho I (conventional type), brand: NEB, catalog number: R0146) restriction endonucleases for double digestion. For the pcDNA3.1 vector plasmid: 15 μL, 10× CutSmart Buffer (brand: New England Biolabs (NEB), product name: rCutSmart™ Buffer (10×), catalog number: B6004V): 2 μL, BamH I (10 U / μL): 1 μL, Xho I (10 U / μL): 1 μL, total volume: 20 μL. Incubate in a water bath at 37°C for 4 hours, inactivate at 65°C for 15 min, and then perform nucleic acid electrophoresis and gel recovery according to the above method.

[0061] Ligate the gel recovery product of the vector and the PCR gel recovery product. For the pcDNA3.1 digested linear vector: 1 μL, the PCR product recovery product: 4 μL, Solution I (brand: TaKaRa, product: DNA Ligation Kit Ver.2.1, catalog number: 6022): 5 μL. After premixing the ligation system on ice, place it in a PCR instrument and react at 16°C for 4 h. Transform the ligation product into DH5α competent cells, coat it on an LB plate containing ampicillin, and culture overnight at 37°C. Pick single colonies and perform PCR identification using universal vector primers (CMV-F: 5′-CGCAAATGGGCGGTAGGCGTG-3′; BGH-R: 5′-TAGAAGGCACAGTCGAGG-3′). Positive clones identified by PCR are subjected to plasmid extraction to remove endotoxins according to the instruction manual of the (QIAGEN, EndoFree Plasmid Mini Kit, catalog number: 12125) kit, and 66 pairs of light and heavy chain plasmids are obtained.

[0062] 4.2 Screening of candidate antibody clones with paired light and heavy chains.

[0063] (1) The paired light and heavy chain plasmids obtained in step 4.1 above were simultaneously transfected into single-well CHO cells in a deep-well plate according to the kit instructions (Thermo Fisher (Gibco) ExpiFectamine™ CHO Kit, catalog number: A29130) for cell culture.

[0064] (2) After 5 days of cell growth, the cell supernatant was collected.

[0065] (3) ELISA detection of cell supernatant, the method is the same as serum ELISA detection (refer to 3.1 Immunization and titer determination of mice). The arrangement of clones obtained after transfecting CHO cells with 66 pairs of light and heavy chain plasmids is shown in Table 3 below.

[0066] Table 3. Ranking list of candidate antibody clones with paired light and heavy chains. A Clone 1 Clones 9 Clones 17 Clones 25 Clones 33 Clones 41 Clones 49 Clones 57 Clone 65 PBS B Clone 2 Clones 10 Clones 18 Clones 26 Clones 34 Clones 42 Clones 50 Clones 58 Clones 66 PBS C Clone 3 Clones 11 Clones 19 Clones 27 Clones 35 Clones 43 Clones 51 Clones 59 Blank cells PBS D Clone 4 Clones 12 Clones 20 Clones 28 Clones 36 Clones 44 Clones 52 Cloning 60 Blank cells PBS E Clones 5 Clones 13 Clones 21 Clones 29 Clones 37 Clones 45 Clones 53 Clones 61 Blank cells mouse positive serum F Clones 6 Clones 14 Clones 22 Clones 30 Clones 38 Clones 46 Clones 54 Clone 62 Blank cells mouse positive serum G Clones 7 Clones 15 Clones 23 Clones 31 Clones 39 Clones 47 Clones 55 Clone 63 Blank cells mouse positive serum H Clones 8 Clones 16 Clones 24 Clones 32 Clones 40 Clones 48 Clones 56 Clone 64 Blank cells mouse positive serum Table 4. Screening results of candidate antibody clones with paired light and heavy chains. B 0.0543 0.0589 0.0474 0.0497 0.0480 0.0504 1.0460 0.0482 0.2128 0.0517 C 0.0520 0.0502 0.3674 0.0482 0.0458 0.0495 0.9055 0.0468 0.0471 0.0477 D 0.0518 0.0510 0.0488 0.0475 0.0469 0.0462 0.0456 0.0452 0.0465 0.0464 E 0.0600 0.0557 0.0531 0.0469 0.7829 0.0494 0.0467 0.0453 0.0457 1.4378 F 0.0535 0.0506 0.0455 0.0450 0.0452 0.0461 1.0314 0.9974 0.0462 1.4759 G 0.0540 0.0478 0.0488 0.0463 0.2051 0.9262 0.8732 0.0456 0.0460 1.4461 H 0.0576 0.0519 0.5700 0.0530 0.0493 0.0497 0.0484 0.0474 0.0473 1.5749 Table 4 shows the detection values ​​of the corresponding clones in Table 3. From Tables 3 and 4, it can be analyzed that clones 19, 24, 37, 39, 47, 50, 51, 54, 55, 62, and 65 are positive clones, for a total of 11 positive clones.

[0067] 4.3 Obtaining the variable region base sequence of candidate positive antibody clones with light and heavy chain pairing.

[0068] According to the ELISA results in Table 4, clone 65 had the highest antibody titer. The plasmid of candidate antibody clone 65 with paired light and heavy chains was sequenced using universal vector primers (CMV-F: 5′-CGCAAATGGGCGGTAGGCGTG-3′; BGH-R: 5′-TAGAAGGCACAGTCGAGG-3′) to obtain the base sequences of the variable regions of the light and heavy chains. The antibody in clone 65 was designated as "ORF4 monoclonal antibody".

[0069] The amino acid sequence of the variable region of the heavy chain of the ORF4 monoclonal antibody is (SEQ ID NO:23): MGWSCIILFLVATGTGVHSQVQLQQPGAELVRPGASVKLSCKASVYTFTSYWMSWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCDRYDDTVDYWGQGTSVTVSS. The amino acid sequence of the light chain variable region is (SEQ ID NO:24): MMSPAQFLFLLVLWIRETNGDVVLTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTNLEIK.

[0070] The nucleotide sequence of the heavy chain variable region of the ORF4 monoclonal antibody is shown in SEQ ID NO:25, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:26.

[0071] Example 5: Large-scale in vitro preparation of ORF4 monoclonal antibody.

[0072] 5.1 In vitro expression of antibodies.

[0073] Pairs of light and heavy chain plasmids from 65 clones were transfected into CHO cells. Transfection was performed simultaneously according to the kit instructions (Thermo Fisher (Gibco) ExpiFectamine™ CHO Kit, catalog number: A29130). Seven days post-transfection, cell cultures were harvested, centrifuged, and the supernatant was collected. Antibody expression was detected by electrophoresis. The results are shown below. Figure 9 As shown: Lane M is the marker, Lane 1 is the supernatant after centrifugation, and Lane 2 is the supernatant after centrifugation of blank cells. The presence of a specific band in Lane 1 indicates successful antibody expression.

[0074] 5.2 Antibody purification.

[0075] (1) After centrifuging the cell supernatant from 5.1, filter it through a 0.45 μm filter membrane.

[0076] (2) Equilibrate the Protein A pre-packed column with 15 column volumes of 0.01M PBS (pH 7.4) buffer.

[0077] (3) Load the supernatant after filtration directly onto the pre-packed Protein A column at low temperature throughout.

[0078] (4) After loading the sample, rinse with 0.01M PBS (pH 7.4) equilibration buffer for 15 column volumes.

[0079] (5) After equilibration, eluent is 0.1M Glycine, pH=2.7. After elution, neutralize immediately with 1M Tris and 1.5M NaCl, pH=8.5.

[0080] (6) Electrophoresis was used to detect the antibody purification results. The results are as follows: Figure 9 As shown: Lane M is the marker, Lane 3 is the first elution with 0.1M glycine, and Lane 4 is the second elution with 0.1M glycine. This result indicates that the ORF4 monoclonal antibody was successfully purified.

[0081] Example 6: Application and efficacy verification of ORF4 monoclonal antibody.

[0082] 6.1 Detection of specific binding of ORF4 monoclonal antibody to ORF4 protein.

[0083] Using ORF4 protein as the antigen and the ORF4 monoclonal antibody (clone 65) screened in Example 5 as the primary antibody, Western blot analysis was performed. The results are as follows: Figure 10 As shown: the bands are clear and free of impurities, indicating that the prepared ORF4 monoclonal antibody has good specificity and strong binding ability to ORF4 protein.

[0084] This result also shows that the ORF4 monoclonal antibody (clone 65) screened in Example 5 can be used as an antibody for Western blot detection and further used for the detection of duck reproductive disorder syndrome virus.

[0085] 6.2 Detection of specific binding of ORF4 monoclonal antibody to ORF4 protein.

[0086] Construction of the eukaryotic expression plasmid pcDNA3.1-ORF4 in a eukaryotic expression system: ORF4 The sequence fragment (SEQ ID NO:5) was ligated into the backbone vector pcDNA3.1+ plasmid (Sangon Biotech, NO. A339023), transformed and purified to obtain the recombinant plasmid pcDNA3.1-ORF4 with correct sequencing. The plasmid was extracted using the EZNA® Endo-Free Mini D6950 endotoxin-free plasmid extraction kit, and the concentration was measured.

[0087] Experimental groups: positive group transfected with pcDNA3.1-ORF4 eukaryotic expression plasmid, negative group transfected with pcDNA3.1+ plasmid.

[0088] Using Lipofectamine 2000 (Thermo Fisher, Cat# 11668-019) as the transfection reagent, transfection was performed according to the following steps: (1) When the 293T cells in the 24-well plate reached a density of 90%, 0.5 μg of pcDNA3.1-ORF4 eukaryotic expression plasmid / pcDNA3.1+ plasmid was added to 50 μL with Opti-MEM (Opti-MEM® I Reduced Serum Medium (Thermo Fisher, Cat# 31985-062)), gently mixed, and incubated at room temperature for 5 min. (2) After gently mixing the reagent, 1.0 μL of Lipofectamine 2000 was added to 50 μL with Opti-MEM, gently mixed, and incubated at room temperature for 5 min. (3) Mix the diluted plasmid DNA with Lipofectamine 2000 at a 1:1 (v / v) ratio (total volume 100 μL), gently invert to mix, and incubate at room temperature in the dark for 20 min (the solution may be slightly turbid, which does not affect transfection). (4) Add 100 μL of DNA liposome complex dropwise to the wells containing 500 μL of antibiotic-free complete medium, and gently shake the culture plate back and forth to mix. (5) Incubate at 37 ℃ in a 5% CO2 incubator. Replace with fresh complete medium 48 hours after transfection, 4–6 h later.

[0089] After transfection, IFA detection was performed according to the following steps: (1) Wash cells with PBS 3 times, 3 min each time; (2) Fix with 4% paraformaldehyde for 15 min, wash with PBS 3 times, 5 min each time; (3) Permeate with 0.5% Triton X-100 (PBS) at room temperature for 15 min, then wash with PBS 3 times, 5 min each time; (4) Blot dry PBS with absorbent paper, block with 5% skim milk prepared with PBS, 37℃, 2 h; (5) Blot dry skim milk with absorbent paper, add 1:1000 diluted ORF4 monoclonal antibody (clone 65) to both positive and negative groups, incubate overnight at 4℃; (6) Then wash with PBST 5 times, 5 min each time, add FITC-labeled fluorescent anti-mouse secondary antibody [SeraCare KPL, rabbit anti-mouse IgG] diluted with PBS in a dark place. [(H+L)-FITC, catalog number 5230-0311], incubated at 37°C for 45 min, then washed three times with PBST for 5 min each time (avoiding light), and the results were then observed using a fluorescence microscope. Results are as follows: Figure 11 As shown: Figure 11The left image shows the positive control, and the right image shows the negative control. The comparison reveals that the positive control exhibits green fluorescence, while the negative control does not. This indicates that the ORF4 monoclonal antibody can specifically bind to the ORF4 protein of duck reproductive disorder virus.

[0090] 6.3. Detection of specific binding of ORF4 monoclonal antibody to duck reproductive disorder syndrome virus.

[0091] The positive Muscovy duck oviduct epithelial primary cells obtained in step S6 of Example 1 were cultured to a density of 90% as a positive control, while negative Muscovy duck oviduct epithelial primary cells were set up as a negative control.

[0092] IFA detection was performed according to the following steps: (1) Wash cells with PBS 3 times, 3 min each time; (2) Fix with 4% paraformaldehyde for 15 min, wash with PBS 3 times, 5 min each time; (3) Permeate with 0.5% Triton X-100 (PBS) at room temperature for 15 min, then wash with PBS 3 times, 5 min each time; (4) Blot dry PBS with absorbent paper, block with 5% skim milk prepared with PBS, 37℃, 2 h; (5) Blot dry skim milk with absorbent paper, add 1:1000 diluted ORF4 monoclonal antibody (clone 65), incubate overnight at 4℃; (6) Then wash with PBST 5 times, 5 min each time, add FITC-labeled fluorescent anti-mouse secondary antibody [SeraCare KPL, rabbit anti-mouse IgG] diluted with PBS in a dark place. [(H+L)-FITC, catalog number 5230-0311], incubated at 37°C for 45 min, then washed three times with PBST for 5 min each time (avoiding light), and the results were then observed using a fluorescence microscope. Results are as follows: Figure 12 As shown: Figure 12 The left image shows the positive control, and the right image shows the negative control. The comparison reveals that the positive control exhibits green fluorescence, while the negative control does not. This indicates that the ORF4 monoclonal antibody can specifically bind to duck reproductive disorder syndrome virus.

[0093] In summary, the prepared ORF4 monoclonal antibody exhibits good specificity and strong binding ability, enabling it to bind to natural viruses. It can be applied in various laboratory settings, including the prevention and treatment of duck reproductive disorder syndrome virus (DRS). It can also be used to prepare DRS virus detection kits or drugs for the prevention and treatment of DRS, demonstrating promising application prospects.

[0094]

[0095] ORF1 nucleotide sequence (SEQ ID NO:2):

[0096] ORF2 nucleotide sequence (SEQ ID NO:3):

[0097] ORF3 nucleotide sequence (SEQ ID NO:4):

[0098] Nucleotide sequence of ORF4 (SEQ ID NO:5): ATGAATCCAAGTGCTCCAGTGTGGACACCGCAGAATAGTAATGGTAATAGTGTTGATGGTCCTCCGTATGGTTATACTAATCCACCGTTTCAGGCTTACCCTTGGGGTAATTTACCATCTAATCGGAATTGGTATCCCAGAAGAGGATGGTGTAGACCATGGCGTGGTCGTGGCAACTGGAATAATAGGAATCAAGACCGCGGGAGATCTCGTGATCGCGCATCATCAAGAGCTCGTTCGAGGAGTCGATCTGCTAGTCGTCGGCGTGGACATGGAGACGACTGGTACAGCCAGTGGGGACACAATGTCGTAACGTATGGTATGGTAACTGTGACACAGCAAGGGAAGTCTAATTTTAATCTTAGGAATCCCTGGAATTATAATAATAAGGACGTTGAACAGACAAAGTTGAGAGACTGGAGATTTCCAGCATCCACTGAAGTTGAAACAGTTGTCTTTGATGGTGATAACAATGGTGGTTTCTATCGTGTTGGGTTGAAAGTTGATATTGCTTCTCCTGATTCGAGGAAAGTAAGATTTGGTACACCTACTGATTTGGCAGATGGTGTTAAAAATTTAACTCTTGATTCTGAAGGCGATAAGAAGAAAAAGCGTGGTGGTATCTTTAGGAGATGGCGTTCCACGGAGCAGTTGTGA。

[0099] Nucleotide sequence of ORF5 (SEQ ID NO:6): ATGATGATCACCATCAATGCACACAACACACTTCGTTTGTTCCTCACAACACAACACCTTACAACCCATACGACCACGATTGCGATCAACAAGTGTTTTGCCAAACCAATCATCGATTTTATTATTACACTTCTTACAACATCTCTCTTTTGCATATTTACAGTCACGAACACTGTGACCAACAACGTTACACCAAATACAGGCTTGGCCAATACAAACATCACAATCACAATTCTTATCACACTGACACCTTGGACAATGACAATCACCATTCAAATTCGAATGTTTACTAATGCCACCACCAACAAACAATTGTTTTTTATCAAATTCACTACTAGATGGCTTAACCTTAGACTCCTGCCTTCTCCGCTGAGGCAAGGCTTCAACAACATCCTGCCTCCTCCGCTGTGGCAAAACTTCAACAACATTTTCCTCAACCGGTAA。

[0100] ORF6 nucleotide sequence (SEQ ID NO:7): ATGCATAACAGCATTATCCAATTCCAAAATGCTCCATCTCTCTTGACCTTAGCAACAAGAGGTCCACGAAAAATTTTAACAACCTTACCATCACAAAGACATGACAACAACCTCTCTCTAACCTCAACAAAACACAACAAAGCCGGATTAATTACTACCACCACCCACGACACAGGTGTTGCAATCCACGGAACAAAAAGCAAAACCAAAATCATAACACAAAACCAAATTCCATTAATAATACCAACATCCATAACACACCAAAACACAAAGACACCAACAAGTGATTTCACATCTCTCATGCCTCGCATATGA。

[0101] ORF7 nucleotide sequence (SEQ ID NO:8): ATGACAAGCCATGATGATCACCATCAATGCACACAACACACTTCGTTTGTTCCTCACAACACAACACCTTACAACCCATACGACCACGATTGCGATCAACAAGTGTTTTGCCAAACCAATCATCGATTTTATTATTACACTTCTTACAACATCTCTCTTTTGCATATTTACAGTCACGAACACTGTGACCAACAACGTTACACCAAATACAGGCTTGGCCAATACAAACATCACAATCACAATTCTTATCACACTGACACCTTGGACAATGACAATCACCATTCAAATTCGAATGTTTACTAA。

[0102] Amino acid sequence of ORF1 (SEQ ID NO:9):

[0103] Amino acid sequence of ORF2 (SEQ ID NO:10): MAFHGAVVICLGFLNLCYCDPFLNFGFSLGWFNDTKVNGSNDSNVTTTSLSTAFGSIQGSVWGVVFGNQDSSGVVYVDWAGRTGGFVGADQIFNFTGNHSALGCNWSSNDTLVAGGPRPPGTRVPQFDNVTVDCGDLAVRLDFGGGSKLICGIDSKEDRHRGQVIGGITFGCDDSGCSSTSYYFRCYGNRSMEIDSNCTNGGGNNTNGTRCGKPVVKRWSFVRPVEEVSDKWNFTDKIVVCSYDCWGFSCADFNNCTCTGGPGAICCSTGNNMTSCRVLGDGTETWWSHHFEVIGLGLIVFLILVICVEIVLFCCGFKRKKKRRWLITNGETTIEAKGWFFVFLLWYCGFRSATAISIGKNDLNRWFSGPEDFGFKVTSLGFVANGSKFEERCCQWGHSTAGGISCAVDTPDNDNDPCYKWSQDPTQWDCRSYENGVAGWNDGCFLENGNTVSWCCKCRCRSSWTGWRFKFTGKPVGSIMLRSANHTLVVNISGDGTYYTAFANVTVSGCTGPMALDAWVSRGHWLDAHSCGLSIGDRPLHGAVHCSGRSNWGSVVTSCYCPQGGELGPISVDVDLGFPYKQKNVPAARVSGTCDVNFEWVEGKQNLGNCSLWCGDWSDGWCQVHIMNDTCIFQNVTQTKGITWIYCGNDMIIGKSKCNGTVWRPTVPGPYNPDEKHDSEHDNGFAHLWKEFLEFWQKWFYWIVIVIIVFVFWLIVRLLFCCNRK。

[0104] Amino acid sequence of ORF3 (SEQ ID NO:11): MIFEWDKDFVKNFYKYLEDKMVKCCGMVNNFPHSIFNRDVVHGLRYLIDNDCNERVVDEFKPGNGFEWLSTGKYVRESGYVYGEYTTRDYIEDMFLKRFYESPFYPELRFNKLSKDFGVKYCRGLISVGMMPSRIRDVCKVNYPFEAEVPSVDLEAERFFKNYDWCNKIIGAYSWPKICLKSEMESVARFCPDERLCSGLEGLKFNSILIAFNYCVEDWNMGRSSVGLSDEDFVVTNRSWTNSPGYPYNKNFCRQAREAFDVYFGLLKHYEYSSMNDWMPTVFNVFCKKEILKAEKVLANDIRTIIAPALCQQLVMQKLTLAISMRVSSNWRFSHTSIGRTRWKDDVDFTGCRIGRFDFICEYDISKWDRSIKSFLLKLFWFYCWFVIDTTKLEHFWQLSNGFESVIYSFMLHKNGEVIRKHYGVPSGFTLTSYVNSWVHTFLNVLCFWELCPVECDINYFKKHCDFVCYGDDGLMGISKEAAEWFTIDARSEWLKTKWGITMDPKKCKMVDRYYFTFDGVDVDGIMFLGDVMKVDEDMGRIVPVFKISKIINQFILGNAVKNYMPSEKILICFGHYVECFFHPNCEVIREYLCFLMNKYKYSYIMKTMFKSEYVEYLAMTNNDLICKIRSLCFDRDKFRAFIMNMFYSVKDESKCSSVDTAE。

[0105] Amino acid sequence of ORF4 (SEQ ID NO:12): MNPSAPVWTPQNSNGNSVDGPPYGYTNPPFQAYPWGNLPSNRNWYPRRGWCRPWRGRGNWNNRNQDRGRSRDRASSRARSRSRSASRRRGHGDDWYSQWGHNVVTYGMVTVTQQGKSNFNLRNPWNYNNKDVEQTKLRDWRFPASTEVETVVFDGDNNGGFYRVGLKVDIASPDSRKVRFGTPTDLADGVKNLTLDSEGDKKKKRGGIFRRWRSTEQL。

[0106] Amino acid sequence of ORF5 (SEQ ID NO:13): MMITINAHNTLRRLFLTTQHLTTHTTTIAINKCFAKPIIDFIITLLTTSLFCIFTVTNTVTNNVTPNTGLA NNTNITITILITLTPWTMTITIQIRMFTNATTNKQLFFIKFTTRWLNLRLLPSPLRQGFNNILPPPLWQNF NNIFLNR.

[0107] ORF6 amino acid sequence (SEQ ID NO:14): MHNSIIQFQNAPSLLTLATRGPRKILTTLPSQRHDNNLSLTSTKHNKAGLITTTTHDTGVAIHGTKSKTKIITQNQIPLIIPTSITHQNTKTPTSDFTSLMPRI.

[0108] ORF7 amino acid sequence (SEQ ID NO:15): MTSHDDHHQCTQHTSFVPHNTTPYNPYDHDCDQQVFCQTNHRFYYYTSYNISLLHIYSHEHCDQQRYTKYRLGQYKHHNHNSYHTDTLDNDNHHSNSNVY.

[0109] ORF1 codon-optimized nucleotide sequence (SEQ ID NO:16):

[0110] nucleotide sequence after codon optimization for ORF2 (SEQ ID NO:17):

[0111] nucleotide sequence optimized for ORF3 codon (SEQ ID NO:18):

[0112] Nucleotide sequence of optimized ORF4 codons (SEQ ID NO:19): ATGAACCCGAGCGCGCCGGTTTGGACCCCGCAGAACAGCAACGGCAACTCTGTTGATGGCCCGCCGTACGGCTACACCAACCCGCCGTTCCAGGCGTACCCGTGGGGCAACCTGCCGTCTAACCGTAACTGGTACCCGCGTCGTGGCTGGTGCCGTCCGTGGCGTGGCCGTGGCAACTGGAACAACCGTAACCAGGATCGTGGCCGTAGCCGTGATCGTGCGAGCAGCCGTGCGCGCAGCCGTTCTCGCAGCGCGAGCCGTCGTCGTGGCCACGGCGATGATTGGTACAGCCAGTGGGGCCACAACGTTGTTACCTACGGTATGGTTACCGTTACCCAGCAGGGCAAAAGCAACTTCAACCTGCGTAACCCGTGGAACTACAACAACAAAGACGTGGAACAGACCAAACTGCGCGACTGGCGCTTCCCGGCGTCCACCGAAGTGGAAACCGTGGTGTTCGATGGCGACAACAACGGCGGCTTCTACCGTGTCGGTCTGAAAGTTGACATCGCTTCTCCGGATAGCCGGAAAGTGCGTTTTGGTACGCCGACCGATCTGGCGGACGGCGTGAAAAACCTGACCCTGGATTCTGAAGGTGATAAGAAGAAAAAACGCGGTGGTATTTTCCGTCGTTGGCGTTCCACCGAGCAGCTGTAA。

[0113] Nucleotide sequence of optimized ORF5 codons (SEQ ID NO:20): ATGATGATCACCATCAACGCTCACAACACCCTGCGTCTGTTCCTGACCACCCAGCATCTGACCACTCACACCACCACCATTGCTATTAACAAATGTTTCGCTAAACCGATTATTGATTTTATTATTACCCTGCTGACTACCTCTCTGTTCTGCATTTTCACTGTTACCAACACCGTTACCAACAACGTTACCCCGAACACCGGTCTGGCGAACACCAACATTACCATTACCATCCTGATTACCCTGACTCCGTGGACCATGACCATTACCATTCAGATTCGTATGTTTACCAACGCTACCACCAACAAACAGCTGTTCTTCATTAAATTCACCACCCGTTGGCTGAACCTGCGTCTGCTGCCGTCTCCGCTGCGTCAGGGTTTTAACAACATCCTGCCGCCGCCGCTGTGGCAGAACTTCAACAACATTTTCCTGAACCGTTAA。

[0114] Optimized nucleotide sequence of ORF6 (SEQ ID NO:21): ATGCACAACAGCATCATCCAGTTTCAGAACGCGCCGAGCCTGCTGACCCTGGCGACCCGTGGCCCGCGTAAAATCCTGACCACCCTGCCGAGCCAGCGACACGATAACAACCTGAGCCTGACCAGCACCAAACACAACAAAGCAGGCCTGATCACCACCACCACCCACGATACCGGAGTTGCGATCCACGGCACCAAAAGCAAAACCAAAATCATCACCCAGAACCAGATCCCGCTGATCATCCCGACCAGCATCACCCACCAGAACACCAAAACCCCGACCAGCGACTTCACCAGCCTGATGCCGCGCATTTAA。

[0115] Optimized nucleotide sequence of ORF7 (SEQ ID NO:22): ATGACCAGCCACGATGACCACCACCAGTGCACCCAGCACACCAGCTTCGTTCCGCACAATACCACCCCGTACAACCCGTACGACCACGACTGCGATCAGCAGGTTTTCTGCCAGACCAACCATCGCTTCTACTACTATACCAGCTACAACATCAGCCTGCTGCACATCTACAGCCACGAACACTGCGACCAGCAACGTTACACCAAATACCGTCTGGGTCAGTACAAACATCACAATCACAACAGCTATCACACCGATACCCTGGATAACGACAACCACCACAGCAACTCCAACGTTTATTAA。

[0116] Nucleotide sequence of the variable region of the heavy chain of the ORF4 monoclonal antibody (SEQ ID NO:25): ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGGTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGTCTACACCTTCACCAGCTACTGGATGAGCTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAATGGATTGGTATGATTGATCCTTCAGACAGTGAAACTCACTACAATCAAATGTTCAAGGACAAGGCCACATTGACTGTTGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGATAGGTACGACGATACTGTGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

[0117] Nucleotide sequence of the variable region of the light chain of the ORF4 monoclonal antibody (SEQ ID NO:26): ATGATGAGTCCTGCCCAGTTCCTGTTTCTGTTAGTGCTCTGGATTCGGGAAACCAACGGTGATGTTGTGCTGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCGGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCTCAGACGTTCGGTGGAGGCACCAACCTGGAAATCAAA。

Claims

1. Monoclonal antibody against duck reproductive disorder syndrome virus, among which, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is MGWSCIILFLVATGTGVHSQVQLQQPGAELVRPGASVKLSCKASVYTFTSYWMSWVKQRPGQGLEWIGMIDPSDSETHYNQMFKDKATLTVDKSSSTAYMQLSSLTSEDSAVYYCDRYDDTVDYWGQGTSVTVSS, and the amino acid sequence of the light chain variable region is MMSPAQFLFLLVLWIRETNGDVVLTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPQTFGGGTNLEIK.

2. The duck reproductive disorder syndrome virus monoclonal antibody according to claim 1, wherein, The duck reproductive disorder syndrome virus has the accession number CCTCC NO:V202619, and the monoclonal antibody can specifically bind to the duck reproductive disorder syndrome virus.

3. A nucleotide molecule encoding the monoclonal antibody of claim 1.

4. The use of the monoclonal antibody according to claim 1 or 2 in the preparation of a test kit for duck reproductive syndrome virus or in the preparation of a drug for the prevention and treatment of duck reproductive syndrome virus.

5. A reagent kit, wherein, The kit contains the monoclonal antibody as described in claim 1.

6. A drug, wherein, The drug contains the monoclonal antibody as described in claim 1.

Images