A monoclonal antibody or antigen-binding fragment thereof against the nucleocapsid protein of oya virus and application thereof

Abstract

The application discloses a kind of monoclonal antibody or its antigen-binding fragment of anti Oya virus nucleocapsid protein and application thereof.The monoclonal antibody F3E10 provided in the application has the characteristics such as high purity, high specificity, high affinity and high sensitivity, can specifically recognize the NP protein of OYAV, can be used as the detection antibody of Western blot and indirect immunofluorescence, is used for the qualitative detection of OYAV-NP protein and OYAV, and can realize the visualization marking and tracing of OYAV under microscope in combination with the secondary antibody labeled with fluorescence, is expected to be widely used in the research, detection and prevention and control of OYAV, and has important popularization and application value in the field of novel bunyavirus research.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody technology, and more specifically, to a monoclonal antibody against Oya virus nucleocapsid protein or its antigen-binding fragment and its applications. Background Technology

[0002] Oya virus (OYAV) belongs to the Panbunniviridae family ( Peribunyaviridae ) Genus Orthobuvirus ( Orthobunyavirus The Simbu serogroup (Simbu) is an arbovirus with a wide host range and high seropositivity rate, posing a risk of cross-species transmission. However, current research on OYAV is limited, and effective detection methods and monitoring systems are still lacking, hindering the effective prevention of potential public health risks.

[0003] The OYAV genome consists of three segments of single-stranded negative-sense RNA. The small (S), medium (M), and large (L) segments encode the nucleoprotein (NP), envelope glycoprotein (GP), and RNA-dependent RNA polymerase (RdRp), respectively. The NP protein has a molecular weight of approximately 25 kDa, with flexible arms extending approximately 20 amino acids from its N-terminus and C-terminus. These arms interact with adjacent NPs to form a stable multimeric structure. Within the inner loop region of the tetramer, the positively charged grooves formed by the NPs specifically bind to and protect the viral RNA, preventing its degradation. Compared to other viral proteins, NPs exhibit high conservation, strong immunogenicity, and stable expression, making them a preferred target for diagnostic antigens.

[0004] Monoclonal antibodies are characterized by high purity, good reproducibility, and strong antigen-binding specificity. However, there are currently no reported antibodies that specifically bind to the Oya virus nucleocapsid protein, nor is there a highly efficient and sensitive method for detecting OYAV. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, this invention provides a monoclonal antibody against the nucleocapsid protein of Oya virus or its antigen-binding fragment and its application, which can effectively detect the virus and provide technical support for OYAV prevention and control.

[0006] The first objective of this invention is to provide a monoclonal antibody against the nucleocapsid protein of Oya virus or an antigen-binding fragment thereof.

[0007] A second objective of this invention is to provide the use of the monoclonal antibody or its antigen-binding fragment in the preparation of products for detecting Oya virus.

[0008] A third objective of this invention is to provide a conjugate.

[0009] The fourth objective of this invention is to provide an immunoassay method for detecting Oya virus nucleocapsid protein for non-disease treatment and diagnostic purposes.

[0010] A fifth objective of this invention is to provide a biomaterial for preparing a monoclonal antibody against the nucleocapsid protein of Oya virus or an antigen-binding fragment thereof.

[0011] A sixth object of the present invention is to provide the use of the said biomaterial in the preparation of monoclonal antibodies against Oya virus nucleocapsid protein or antigen-binding fragments thereof.

[0012] To achieve the above objectives, the present invention is implemented through the following solution:

[0013] This invention provides a monoclonal antibody (i.e., F3E10 antibody) against Oya virus nucleocapsid protein (i.e., OYAV-NP) and its applications. This monoclonal antibody has good reactivity, high titer, and strong specificity, and can be used for detection of OYAV-NP protein by Western blot and indirect immunofluorescence, providing necessary technical means for the diagnosis, prevention and treatment of OYAV.

[0014] A monoclonal antibody against the nucleocapsid protein of Oya virus or its antigen-binding fragment thereof, wherein the monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises CDR-F3E10-H1 to CDR-F3E10-H3, wherein the amino acid sequence of CDR-F3E10-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-F3E10-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-F3E10-H3 is shown in SEQ ID NO.4; the light chain variable region comprises CDR-F3E10-L1 to CDR-F3E10-L3, wherein the amino acid sequence of CDR-F3E10-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-F3E10-L2 is KVS, and the amino acid sequence of CDR-F3E10-L3 is shown in SEQ ID NO.11.

[0015] Preferably, the heavy chain variable region further comprises FR-F3E10-H1 to FR-F3E10-H4, wherein the amino acid sequence of FR-F3E10-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-F3E10-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-F3E10-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-F6-H4 is shown in SEQ ID NO.8.

[0016] In some specific embodiments, the amino acid sequence of the heavy chain variable region has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO.1.

[0017] More preferably, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1.

[0018] Preferably, the light chain variable region further comprises FR-F3E10-L1 to FR-F3E10-L4, wherein the amino acid sequence of FR-F3E10-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-F3E10-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-F3E10-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-F3E10-L4 is shown in SEQ ID NO.15.

[0019] In some specific embodiments, the amino acid sequence of the light chain variable region has at least 95%, 96%, 97%, 98%, or 99% similarity to SEQ ID NO.9.

[0020] More preferably, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.9.

[0021] In some specific embodiments, the monoclonal antibody or its antigen-binding fragment further comprises an Fc segment. The present invention does not specifically limit the species origin of the Fc segment, including but not limited to human Fc segments, mouse Fc segments, rabbit Fc segments, or biologically acceptable modifications or mutations thereof.

[0022] In some specific embodiments, the monoclonal antibody or its antigen-binding fragment further includes a heavy chain constant region and a light chain constant region. Preferably, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD; the light chain constant region includes a light chain constant region selected from κ or λ types.

[0023] The use of any of the monoclonal antibodies or their antigen-binding fragments in the preparation of products for detecting Oya virus.

[0024] A conjugate comprising any of the said monoclonal antibodies or their antigen-binding fragments, and a chemical moiety conjugated thereto.

[0025] In some specific embodiments, the chemical portion is attached to the monoclonal antibody or its antigen-binding fragment of the present invention using any number of methods known to those skilled in the art. Covalent and non-covalent attachment methods can be used. The procedure for attaching the chemical portion to the monoclonal antibody or its antigen-binding fragment is tailored to the chemical structure of the chemical portion.

[0026] An immunoassay method for detecting Oya virus nucleocapsid protein for non-disease treatment and diagnostic purposes, comprising performing immunoassay on the test sample using any of the monoclonal antibodies or their antigen-binding fragments.

[0027] The monoclonal antibody or its antigen-binding fragment provided by the present invention can achieve immunoassays including but not limited to flow cytometry, immunofluorescence, ELISA and immunoprecipitation detection methods.

[0028] Preferably, the immunoassay method is Western blotting or indirect immunofluorescence.

[0029] A biological material for preparing a monoclonal antibody against the nucleocapsid protein of Oya virus or an antigen-binding fragment thereof, wherein it is any one of the following (1) to (5):

[0030] (1) A nucleic acid molecule encoding any of the monoclonal antibodies or their antigen-binding fragments;

[0031] (2) An expression cassette containing the nucleic acid molecule described in (1);

[0032] (3) A recombinant vector containing the expression cassette described in (2);

[0033] (4) Host cells containing the recombinant vector in (3);

[0034] (5) A protein expression system comprising the host cell described in (4).

[0035] The present invention does not have any particular limitation on the type of recombinant vector. Any vector capable of expressing the nucleic acid molecule or the expression cassette can achieve the purpose of the present invention.

[0036] This invention does not have any special limitations on the source and type of host cells. Prokaryotic and eukaryotic cells that can conventionally carry the vector, such as Escherichia coli, HEK293 cells, and CHO cells, can all achieve the purpose of this invention.

[0037] The application of the biomaterials in the preparation of monoclonal antibodies against Oya virus nucleocapsid protein or their antigen-binding fragments.

[0038] A method for preparing a monoclonal antibody or antigen-binding fragment thereof against Oya virus nucleocapsid protein, comprising culturing host cells under conditions allowing expression of the monoclonal antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof from the culture of the host cells.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The monoclonal antibody F3E10 provided by this invention has the characteristics of high purity, high specificity, high affinity and high sensitivity. It can specifically recognize the NP protein of OYAV and can be used as a detection antibody for Western blot and indirect immunofluorescence for qualitative detection of OYAV-NP protein and OYAV. Combined with a fluorescently labeled secondary antibody, it can realize the visualization and tracing of OYAV under a microscope. It is expected to be widely used in the research, detection and prevention of OYAV and has important application value in the field of novel Bunyavirus research. Attached Figure Description

[0041] Figure 1 Agarose gel electrophoresis image of the prokaryotic expression vector pET28a-OYAV-NP for PCR identification of OYAV-NP protein; lanes 1-6 correspond to 6 single colonies picked after transformation.

[0042] Figure 2 The image shows the results of Western blot analysis of the OYAV-NP recombinant protein.

[0043] Figure 3 The image shows the results of identifying the expression form of the OYAV-NP recombinant protein.

[0044] Figure 4 The image shows the SDS-PAGE results of OYAV-NP recombinant protein after purification by denaturing nickel column. The values ​​20 mM to 500 nM represent the flow-through collected after elution with the corresponding working concentration of imidazole.

[0045] Figure 5 The image shows the SDS-PAGE results of OYAV-NP recombinant protein after purification by molecular sieve chromatography. Lane NP represents the product purified by molecular sieve chromatography.

[0046] Figure 6 The image shows the SDS-PAGE results of the F3E10 antibody obtained after purification of mouse ascites fluid. Lanes 1-3 represent bovine serum albumin, DTT-free antibody, and DTT antibody, respectively.

[0047] Figure 7The image shows the results of Western blot analysis of A549 cell protein samples from the blank group and the virus-infected group using the F3E10 antibody.

[0048] Figure 8 The fluorescence micrographs of OYAV-infected cells detected by indirect immunofluorescence assay with F3E10 antibody were observed using an EVOSM5000 fluorescence microscope with an objective magnification of 20×. A represents the blank group; B represents the virus-infected group. Detailed Implementation

[0049] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified. The OYAV GZ8H16 strain used in the following embodiments was isolated and preserved by the inventor's research group at Sun Yat-sen University. It is publicly available from the applicant. The L-segment sequence of this strain is numbered PQ463773.1, the M-segment sequence is numbered PQ463774.1, and the S-segment sequence is numbered PQ463775.1 in the NCBI database.

[0050] Example 1: Prokaryotic expression, purification, and identification of OYAV-NP recombinant protein

[0051] 1. Construction of a prokaryotic expression vector for OYAV-NP recombinant protein

[0052] The sequence of the OYAV-NP gene (GenBank accession number: PQ463775.1, 45bp to 746bp) was synthesized and ligated into the pET-28a(+) vector. BamHI site and XhoI Between the sites, a prokaryotic expression vector for the OYAV-NP recombinant protein was obtained, denoted as pET28a-OYAV-NP.

[0053] pET28a-OYAV-NP was transformed into competent E. coli cells ( E. coli DH5α was used for plating culture, and single colonies were picked for PCR identification, with H2O as a negative control. Results are as follows: Figure 1 As shown, all six monoclonal colonies were positive, with a gene fragment size of 800 bp, consistent with expectations. Monoclonal colonies identified as having correct sequences were retained.

[0054] 2. Prokaryotic expression and identification of OYAV-NP recombinant protein

[0055] Transform the correctly sequenced pET28a-OYAV-NP plasmid into competent cells expressing engineered bacteria. E. coli BL21), plating culture, picking single colonies for expansion culture, when OD 600 When the concentration reached 0.6–0.8, 1 mM IPTG was added to the final concentration, and expression was induced for 24 h at 20 °C and 220 rpm. The bacterial culture was collected and recorded as NP culture. The bacterial culture without IPTG was induced for 24 h at 20 °C and 220 rpm as a control and recorded as control culture.

[0056] After centrifuging the NP bacterial culture and the control bacterial culture, the two bacterial cells were collected, and 5×SDS-PAGE loading buffer was added to each. The cells were boiled in a boiling water bath for 10 min, centrifuged again, and the supernatant was used as the electrophoresis sample. Western blot was used to identify whether OYAV-NP was successfully expressed. The antibody used was an anti-His tag antibody (manufacturer: ThermoFisher, catalog number: MA533032). The results are as follows: Figure 2 As shown, the lane of the NP bacterial solution has a specific band with a molecular weight of approximately 27 kDa, while no obvious band was observed in the lane of the control bacterial solution, indicating that the prokaryotic cells transformed with pET28a-OYAV-NP successfully expressed the OYAV-NP recombinant protein.

[0057] The induced bacterial cells were sonicated and the supernatant and inclusion bodies were obtained by centrifugation. The distribution of the target protein was then analyzed using SDS-PAGE and Coomassie Brilliant Blue staining. Figure 3 As shown, both the lanes of the supernatant and the lanes of the inclusion bodies show obvious bands at 27 kDa, indicating that the OYAV-NP recombinant protein can be solublely expressed in the prokaryotic system.

[0058] 3. Purification and identification of OYAV-NP recombinant protein

[0059] (1) Ni column purification

[0060] The supernatant obtained after sonication in the previous step was collected and purified by nickel affinity chromatography. First, the Ni-NTA column was equilibrated with 5 column volumes of Binding Buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 8.0). Then, the supernatant was filtered through a 0.45 μm filter and loaded onto the equilibrated Ni-NTA column at a flow rate of 0.5 mL / min. The column was washed with 10 column volumes of Wash Buffer (20 mM Tris-HCl, 500 mM NaCl, 50 mM imidazole, pH 8.0) to remove contaminating proteins. Elution was then performed sequentially with imidazole elution buffers at working concentrations of 20 mM, 50 mM, 100 mM, 200 mM, and 500 nM, and the flow-through at each concentration was collected.

[0061] The flow-through solutions of various concentrations, the supernatant and inclusion bodies collected after the previous ultrasonic disruption, and the control bacterial culture were analyzed for the distribution of the target protein using SDS-PAGE and Coomassie Brilliant Blue staining. The results are as follows: Figure 4 As shown, most of the impurities in the supernatant collected after ultrasonic disruption were removed by nickel column purification, and the purity of the OYAV-NP recombinant protein reached 60%.

[0062] The flow-through obtained by elution with imidazole at a working concentration of 500 nM was collected and concentrated to 1-2 mL through an ultrafiltration centrifuge tube (10 kDa molecular weight cutoff) to obtain a crude pure solution of OYAV-NP recombinant protein.

[0063] (2) Molecular sieve chromatography purification

[0064] The crude OYAV-NP recombinant protein solution obtained in the previous step was further purified by molecular sieve chromatography and identified by SDS-PAGE. The specific steps are as follows: Column equilibration was performed using molecular sieve buffer (20 mM Tris-HCl, 150 mM NaCl, pH 8.0). The concentrated sample was then filtered through a 0.22 μm filter and loaded onto the column. Finally, isocratic elution was carried out with equilibration buffer, and the target protein fraction was collected based on the A280 absorption peak. The high-purity fractions were then combined. Figure 5 As shown, after purification by molecular sieve chromatography, the crude pure solution of OYAV-NP recombinant protein showed a single band after Coomassie brilliant blue staining, indicating that the purity of OYAV-NP recombinant protein was increased to over 95%, and it can be used as an immunogen for animal immunization.

[0065] Example 2: Preparation, purification, and sequencing of OYAV-NP protein monoclonal antibody

[0066] 1. Animal immunization

[0067] The purified OYAV-NP recombinant protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and emulsified completely. This mixture was then used as an immunogen to immunize four SPF-grade BALB / c female mice. Immunizations were administered at two-week intervals, with immunization sites sequentially: footpads, subcutaneous tissue, and muscle. Human blood was collected from each mouse on day 7 after the fourth immunization.

[0068] 2. Indirect ELISA detection of serum titers in immunized mice

[0069] The collected blood was placed in a 1.5 mL centrifuge tube and incubated at room temperature for 1 h. The separated serum was then pipetted and stored at -20℃. 10 μg of the OYAV-NP recombinant protein obtained in Example 1 was coated into each well of a 96-well microplate and incubated overnight at 4℃. After washing three times with PBST, 100 μL of 5% skim milk was added to each well, and the plate was blocked at 37℃ for 1 h. After washing three times with PBST, the serum was serially diluted with PBST containing 5% FBS (fetal bovine serum), and 100 μL was added to each well, and the plate was incubated at 37℃ for 1 h. Then, 100 μL of 1:2000 diluted HRP-labeled goat anti-mouse IgG was added to each well, and the plate was incubated at 37℃ for 1 h. After washing five times with PBST, 100 μL of TMB chromogenic solution was added to each well, and the plate was incubated at 37℃ for 15 min. Finally, 50 μL of stop solution was added, and the OD value was read using a microplate reader. 450 .

[0070] Table 1. Results of ELISA detection of antiserum titers in four immunized mice.

[0071]

[0072] Note: The potency is the serum dilution factor corresponding to the minimum OD reading that is greater than 2 / 3 of the maximum OD.

[0073] The ELISA results are shown in Table 1. Mice were immunized with purified OYAV-NP recombinant protein. Seven days after the fourth immunization, all four immunized mice (A211, A212, A213, and A214) exhibited a strong humoral immune response, with significantly increased serum antibody levels. Furthermore, the OD value showed a regular decrease with increasing serum dilution, indicating reliable detection results. Using the mean OD value of the blank control + 0.1 as the cutoff value, the serum titer of all mice reached 1:512000, indicating the production of high-titer specific antibodies. Mouse A214 showed the highest OD value across most dilution gradients, suggesting that its serum may have the optimal titer and affinity for specific antibodies. Therefore, A214 mice were selected for subsequent cell fusion experiments.

[0074] 3. Cell fusion

[0075] Spleen cells from A214 mice after booster immunization were fused with SP2 / 0 cells at a ratio of 15:1 in a 37°C water bath. 1 mL of preheated PEG was added dropwise over 45 seconds, with the fusion tube held firmly in the left palm and the right hand, and the tube vigorously shaken up and down. The mixture was allowed to incubate at room temperature for 90 seconds. Then, 1 mL of DMEM medium was added over 30 seconds, followed by 2 mL over 30 seconds, and finally 13 mL over 2 minutes to terminate the PEG incubation. The fusion tube was then incubated at 37°C in a 5% CO2 incubator for 10 minutes, followed by centrifugation at 1000 rpm for 10 minutes. The cells were then plated with 10 mL of HAT complete medium. Once the confluence of fused cells reached over 50%, positive cell lines were selected.

[0076] 4. Hybridoma cell screening

[0077] A549 cells were seeded in 6-well plates. After the cell confluence reached 80%, OYAV GZ8H16 virus solution was added to infect A549 cells at a dose of MOI=1 as the virus infection group. Uninfected A549 cells were used as the control group. The cells were cultured for another 48 hours.

[0078] The culture supernatant of the fusion cells was collected 10 days after fusion and added to the virus-infected group and the blank group, respectively. Western blot and indirect immunofluorescence assays were performed to screen for positive cell clones. The secondary antibody used for Western blot was IRDye®800CW-labeled goat anti-mouse IgG antibody, and the secondary antibody used for indirect immunofluorescence assay was FITC-labeled goat anti-mouse IgG (full molecular weight) antibody. Fusion cells that reacted positively with the virus-infected group and negatively with the blank group were retained to obtain hybridoma cells.

[0079] 5. Hybridoma cell cloning

[0080] Subcloning of hybridoma cells was performed using a limiting dilution method, with the following steps: Mouse spleen cells were used to prepare a feeder layer. Hybridoma cells were aspirated and counted, followed by serial dilutions to ensure a cell count of 100–150 per well. 13 mL of HT medium was prepared, and the liquid from each well was transferred to HT medium and repeatedly pipetted to mix thoroughly. Two drops were then added to each well of a cell culture plate coated with feeder cells and incubated in an incubator. After 3–4 days, the number of cell clones in each well was counted. On days 8–9, the cell supernatant from the single-clone wells was collected, and titer and specificity were determined using the indirect ELISA method described in this embodiment. After four rounds of subcloning and indirect immunofluorescence screening, a hybridoma cell line stably secreting a monoclonal antibody against OYAV-NP protein was finally obtained, designated F3E10 cells, and the secreted monoclonal antibody was designated F3E10 antibody.

[0081] 6. Preparation and purification of ascites fluid

[0082] F3E10 cells were transferred to 24-well plates pre-coated with feeder cells (mouse spleen cells). After the F3E10 cells reached confluence, cells from 8 wells were collected into 15 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in 700 μL of physiological saline. The cells were then injected into the peritoneal cavity of mice, and simultaneously, 500 μL of sterile paraffin oil was injected. The mice's abdomens were massaged to facilitate diffusion within the peritoneal cavity. Excess cells were collected and cryopreserved, with one mouse cryopreserved for every 8 wells. Ascites fluid from the mice was collected and purified by affinity chromatography using MCE HY-K0214 Protein G Agarose to obtain the purified F3E10 antibody.

[0083] The reducing agent dithiothreitol (DTT) breaks the disulfide bonds between antibody chains, breaking the intact antibody into two heavy chains and two light chains. The purified F3E10 antibody is mixed with SDS loading buffer without DTT and boiled for 5 min to obtain an F3E10 antibody sample with a complete dimer structure (denoted as DTT-free antibody). The purified F3E10 antibody is mixed with SDS loading buffer containing DTT and boiled for 5 min to obtain F3E10 antibody in the light and heavy chain form (denoted as DTT antibody).

[0084] The presence of DTT antibody and DTT antibody were identified by SDS-PAGE electrophoresis, with 1.00 μg bovine serum albumin (BSA) used as a negative control. Results are as follows: Figure 6 As shown, the F3E10 antibody without DTT exhibits a single band with a molecular weight of approximately 150 kDa, indicating that the antibody is intact, free of other protein impurities, and successfully purified. The F3E10 antibody with DTT shows two bands: a 55 kDa heavy chain and a 25 kDa light chain, verifying that the antibody is indeed an IgG antibody, indicating successful antibody preparation.

[0085] 7. Sequence analysis of the variable region of the F3E10 antibody

[0086] According to the International Immunogenetic Information System ® (THE INTERNATIONAL IMMUNOGENETICSINFORMATION SYSTEM ® IMGT ® The domains of the F3E10 antibody are defined as follows:

[0087] The amino acid sequence of the heavy chain variable region (VH) is: EVMLVESGGGLVKPGGSLKLSCAASGITVNSYAMSWVRQTPEKRLEWVATISSRGTYTYYPDSVKGRFTISRDIAKDTLYLQMTSLGSEDTAIYYCARLGYYDPYFAMDYWGRGTSVTVSS (SEQ ID NO.1), of which the amino acid sequence of the complementarity-determining region CDR-F3E10-H1 is: GITVNSYA (SEQ ID NO.2); the amino acid sequence of CDR-F3E10-H2 is: ISSRGTYT (SEQ ID NO.3); the amino acid sequence of CDR-F3E10-H3 is: ARLGYYDPYFAMDY (SEQ ID NO.4); and the amino acid sequence of the backbone region FR-F3E10-H1 is: EVMLVESGGGLVKPGGSLKLSCAAS (SEQ ID NO.1). NO.5); The amino acid sequence of FR-F3E10-H2 is: MSWVRQTPEKRLEWVAT (SEQ ID NO.6); The amino acid sequence of FR-F3E10-H3 is: YYPDSVKGRFTISRDIAKDTLYLQMTSLGSEDTAIYYC (SEQ ID NO.7); The amino acid sequence of FR-F3E10-H4 is: WGRGTSVTVSS (SEQ ID NO.8).

[0088] The amino acid sequence of the light chain variable region (VL) is: DVVMTQTPLSLPVSLGDHASISCRSSQSLVHINGNTYLHWYLQKPGQSPNLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVAVTFGAGTKLELK (SEQ ID NO. 9), of which the amino acid sequence of the complementarity-determining region CDR-F3E10-L1 is: QSLVHINGNTY (SEQ ID NO. 10); the amino acid sequence of CDR-F3E10-L2 is: KVS; and the amino acid sequence of CDR-F3E10-L3 is: SQSTHVAVT (SEQ ID NO. 11). The amino acid sequence of the backbone region FR-F3E10-L1 is: DVVMTQTPLSLPVSLGDHASISCRSS (SEQ ID NO. 12); and the amino acid sequence of FR-F3E10-L2 is: LHWYLQKPGQSPNLLIY (SEQ ID NO. 11). NO.13); The amino acid sequence of FR-F3E10-L3 is: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC (SEQ ID NO.14); The amino acid sequence of FR-F3E10-L4 is: FGAGTKLELK (SEQ ID NO.15).

[0089] Example 3: Evaluation of the detection performance of the F3E10 antibody

[0090] 1. Western blot detection performance of F3E10 antibody

[0091] (1) Cell culture and infection

[0092] A549 cells were seeded in 6-well plates. After the cell confluence reached 80%, OYAV GZ8H16 virus solution was added to infect A549 cells at a dose of MOI=1 as the virus infection group. Uninfected A549 cells were used as the control group. The cells were cultured for another 48 h.

[0093] (2) Western blot detection

[0094] Protein samples from two groups of A549 cells were collected, lysed with NP-40 lysis buffer for 30 min and centrifuged. The cell supernatant was collected and 5× loading buffer was added. The mixture was then boiled at 100℃ for 10 min to obtain pretreated protein samples.

[0095] After sample loading and separation by SDS-PAGE electrophoresis, the gel fragment containing the target band was excised and placed in a transfer apparatus, and transferred semi-dry for 50 min at a constant voltage of 20V. The membrane was then blocked with 5% skim milk at room temperature for 2 h. The blocked membrane was washed three times with PBST for 5 min each time. The F3E10 antibody purified in Example 2 was added as the primary antibody (dilution 1:500), and incubated at room temperature for 1.5 h, followed by three washes with PBST for 5 min each time. Subsequently, rabbit anti-mouse IgG secondary antibody labeled with infrared fluorescent dye (dilution 1:10000) was added, and the membrane was incubated at room temperature in the dark for 45 min, followed by three washes with PBST in the dark for 5 min each time. Finally, the results were scanned and observed using an infrared laser scanning imaging system.

[0096] (3) Test results

[0097] like Figure 7 As shown, no band was detected in the blank group, while a band of 26 kDa was detected in the virus-infected group, which is consistent with the size of the NP protein. This indicates that the F3E10 antibody prepared in this invention can undergo a specific immune reaction with the OYAV-NP protein, and the qualitative detection of the OYAV-NP protein by Western blot method has been successfully achieved.

[0098] 2. Indirect fluorescent immunoassay performance of F3E10 antibody

[0099] (1) Cell culture and infection

[0100] A549 cells were seeded in 24-well plates. After the cells reached 90% confluence, OYAV GZ8H2016 virus solution was added to infect A549 cells at an MOI of 1 as the virus infection group, and uninfected A549 cells were used as the control group. After adsorption at 37°C for 2 hours, the culture supernatant of both groups of cells was discarded and replaced with fresh DMEM containing 4% serum. The cells were then cultured at 37°C for another 24 hours.

[0101] (2) Indirect immunofluorescence detection

[0102] Discard the culture medium from both cell groups, wash twice with PBS, and fix with 4% paraformaldehyde at room temperature for 30 min. Then discard the fixative, wash three times with PBS, add permeabilization buffer containing 0.1% Triton X-100, and permeabilize for 15 min at room temperature. Discard the permeabilization buffer, wash three times with PBS, and block with 5% skim emulsion at room temperature for 2 h. Discard the blocking buffer, add the F3E10 antibody purified in Example 2 as the primary antibody (dilution 1:16), and incubate at room temperature for 2 h. Discard the primary antibody, wash three times with PBS, add Alexa Fluor 488 fluorescently labeled donkey anti-mouse IgG (H+L) secondary antibody (dilution 1:200), and incubate at room temperature in the dark for 45 min. Discard the secondary antibody, wash three times with PBS in the dark, 5 min each time. Observe the results using an inverted fluorescence microscope.

[0103] (3) Test results

[0104] like Figure 8 As shown, no fluorescence was detected in the blank group, while the virus-infected group showed a clear and specific green fluorescent signal, indicating that the F3E10 antibody prepared in this invention reacts specifically with the OYAV-NP protein, successfully achieving the qualitative detection of the OYAV-NP protein by indirect immunofluorescence.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A monoclonal antibody against the nucleocapsid protein of Oya virus or its antigen-binding fragment, characterized in that, The monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises CDR-F3E10-H1 to CDR-F3E10-H3, wherein the amino acid sequence of CDR-F3E10-H1 is shown in SEQ ID NO.2, the amino acid sequence of CDR-F3E10-H2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR-F3E10-H3 is shown in SEQ ID NO.4; The light chain variable region comprises CDR-F3E10-L1 to CDR-F3E10-L3, wherein the amino acid sequence of CDR-F3E10-L1 is shown in SEQ ID NO.10, the amino acid sequence of CDR-F3E10-L2 is KVS, and the amino acid sequence of CDR-F3E10-L3 is shown in SEQ ID NO.

11.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region further comprises FR-F3E10-H1 to FR-F3E10-H4, wherein the amino acid sequence of FR-F3E10-H1 is shown in SEQ ID NO.5, the amino acid sequence of FR-F3E10-H2 is shown in SEQ ID NO.6, the amino acid sequence of FR-F3E10-H3 is shown in SEQ ID NO.7, and the amino acid sequence of FR-F3E10-H4 is shown in SEQ ID NO.

8.

3. The monoclonal antibody or its antigen-binding fragment according to claim 2, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

1.

4. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, The light chain variable region further comprises FR-F3E10-L1 to FR-F3E10-L4, wherein the amino acid sequence of FR-F3E10-L1 is shown in SEQ ID NO.12, the amino acid sequence of FR-F3E10-L2 is shown in SEQ ID NO.13, the amino acid sequence of FR-F3E10-L3 is shown in SEQ ID NO.14, and the amino acid sequence of FR-F3E10-L4 is shown in SEQ ID NO.

15.

5. The monoclonal antibody or its antigen-binding fragment according to claim 4, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.

9.

6. The use of the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5 in the preparation of products for detecting Oya virus.

7. A conjugate, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5, and the chemical moiety conjugated thereto.

8. An immunoassay method for detecting Oya virus nucleocapsid protein for non-disease treatment and diagnostic purposes, characterized in that, Immunoassay was performed on the test sample using the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5.

9. A biomaterial for preparing a monoclonal antibody against the Oya virus nucleocapsid protein or an antigen-binding fragment thereof, characterized in that, It is any one of the following (1) to (5): (1) A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 5; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the expression cassette described in (2); (4) Host cells containing the recombinant vector in (3); (5) A protein expression system comprising the host cell described in (4).

10. The use of the biomaterial of claim 9 in the preparation of monoclonal antibodies against Oya virus nucleocapsid protein or antigen-binding fragments thereof.

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