Hybridoma cell lines secreting monoclonal antibodies against cyprinid herpesvirus type 3 IL-10, monoclonal antibodies and their applications

Abstract

This invention discloses a hybridoma cell line that secretes a monoclonal antibody against cyprinid herpesvirus type 3 (CyHV) IL-10, the monoclonal antibody, and its applications. The hybridoma cell line CyHV31-IL10 was deposited on August 17, 2022, at the China Center for Type Culture Collection (CCTCC) in Wuhan, Hubei Province, with accession number CCTCC C2022212. The monoclonal antibody secreted by CyHV3-IL-10 can recognize the prokaryotic expression product, eukaryotic expression product, and extracellular functional region protein of CyHV3-IL-10. It is used for the immunological detection of CyHV-3-IL-10, providing parameters for the diagnosis, treatment, or prevention of Cyprinid herpesvirus type 3 infection, and has potential application value in fish disease control.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, specifically relating to a hybridoma cell line that secretes monoclonal antibodies against cyprinid herpesvirus type 3 IL-10, the monoclonal antibody, and its applications. Background Technology

[0002] Carp (Cyprinuscarpio) is one of the most affordable and high-quality sources of protein for humans, and increasing carp production could help address global food issues to some extent. Koi, with their vibrant colors and graceful movements, are among the most prized ornamental fish and are beloved by pet enthusiasts. Koi herpesvirus disease (KHVD) causes mass mortality in carp, koi, and their variants, with mortality rates exceeding 90% during large-scale outbreaks. Its high morbidity and mortality rates result in severe economic losses to the aquaculture industry. Koi herpesvirus (KHV), also known as Cyprinuscarpio Herpesvirus 3 (CyHV-3), is the causative agent of koi herpesvirus disease. CyHV-3, belonging to the family Heteroherpesviridae, order Herpesvirales, and genus Herpesvirus, is highly infectious and has a wide host range, but it only induces disease in carp and koi. Infected fish exhibit typical clinical symptoms of kidney hemorrhage and swelling, and gill necrosis. CyHV-3 is a double-stranded DNA virus with a genome size of 295 bp, encoding 164 open reading frames (ORFs), of which ORF134 encodes viral interleukin-10.

[0003] Th2 cells can assist B cells in differentiating into antibody-secreting cells and participate in humoral immune responses. Although the two cell types secrete different cytokines, there is an interactive regulatory effect. In 1989, Fiorentino et al. in the United States discovered that mouse Th2 cells can produce a new cytokine that can inhibit the transcription of Th1 cytokine mRNA. Based on its function, it was named cytokine synthesis inhibitory factor (CSIF) and in the same year, it was named interleukin-10 (IL-10). Studies have shown that IL-10 is a cytokine with multi-directional regulatory functions. Abnormal IL-10 can cause immune dysfunction and some pathological damage. In addition to its important immunomodulatory activity, IL-10 is also related to the occurrence and development of various immune diseases.

[0004] To date, cyprinid herpesvirus type 3 (CyHV-2) can be isolated and identified using cell culture, serum neutralization assays, molecular biology, ELISA, electron microscopy, and in situ hybridization. Studies have reported the preparation and identification of polyclonal antibodies against the protein encoded by the ORF136 gene of CyHV-3: first, ORF136 is cloned and ligated into a vector for prokaryotic protein expression and purification. The purified recombinant protein is then injected subcutaneously into rabbits at multiple sites, purified using affinity purification of antiserum, and its specificity is determined by SDS-PAGE, Western blotting, and indirect immunofluorescence. Other studies have established an immunological detection method for the capsid protein ORF72 of CyHV-2, using purified recombinant ORF72 protein as the detection antigen. Clinical sample testing has shown the ability to detect ORF72-specific antibodies.

[0005] Existing technologies have varying degrees of specificity in obtaining recombinant proteins and in screening polyclonal and monoclonal antibodies after protein purification. It is difficult to obtain recombinant proteins to cultivate monoclonal hybridoma cell lines and monoclonal antibodies cannot be prepared. Currently, there are no reports on screening specific hybridoma cell lines after obtaining recombinant proteins and using them to prepare monoclonal antibodies against cyprinid herpesvirus type 3 IL-10. Summary of the Invention

[0006] The first objective of this invention is to provide a recombinant protein of cyprinid herpesvirus type 3 IL-10, which is obtained by a preparation method comprising the following steps: the open reading frame sequence of cyprinid herpesvirus type 3 IL-10 is shown in SEQ ID NO:1, and the amino acid sequence encoded by it is shown in SEQ ID NO:2. It is ligated into the expression vector pET-21d(+), which is then transformed into competent cells of expression strain Rosetta(DE3). After IPTG induction, the cells are centrifuged, and the collected cells are cyclically lysed under ultra-low temperature and high pressure for 8–12 min. Inclusion bodies are extracted and renatured at 4°C for 36 h using the dilution renaturation method. The renaturation solution is concentrated and replaced with protein buffer, then centrifuged at high speed, and the supernatant is purified to obtain the recombinant protein of cyprinid herpesvirus type 3 IL-10.

[0007] A second objective of this invention is to provide the application of the above-mentioned recombinant IL-10 protein of cyprinid herpesvirus type 3 in the preparation of molecular immune adjuvants.

[0008] A third objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against cyprinid herpesvirus type 3 IL-10. This hybridoma cell line, CyHV31-IL10 (Cyprinuscarpio HerpesVirus31-IL10), was deposited on August 17, 2022, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCC C2022212.

[0009] Preferably, the hybridoma cell line that secretes monoclonal antibodies against cyprinid herpesvirus type 3 IL-10 is obtained by screening for the recombinant protein of cyprinid herpesvirus type 3 IL-10.

[0010] A fourth objective of this invention is to provide the use of the hybridoma cell line that secretes monoclonal antibodies against cyprinid herpesvirus type 3 IL-10 in the preparation of reagents for diagnosing or detecting cyprinid herpesvirus type 3 infection.

[0011] The fifth objective of this invention is to provide a monoclonal antibody against cyprinid herpesvirus type 3 IL-10, which is obtained by secretion from the hybridoma cell line and has the function of specifically recognizing cyprinid herpesvirus type 3.

[0012] The sixth objective of this invention is to provide the application of the above-mentioned anti-cyprinid herpesvirus type 3 IL-10 monoclonal antibody in the preparation of reagents for diagnosing or detecting cyprinid herpesvirus type 3 infection.

[0013] A seventh objective of the present invention is to provide a kit for the immunoassay of cyprinid herpesvirus type 3, comprising the aforementioned anti-cyprinid herpesvirus type 3 IL-10 monoclonal antibody.

[0014] The eighth objective of this invention is to provide the above-mentioned kit for detecting the concentration of cyprinid herpesvirus type 3 IL-10 in carp blood or body fluids or in carp tissue sections.

[0015] In some technical solutions of this invention, firstly, the open reading frame sequence (with the coding signal peptide sequence removed) of cyprinid herpesvirus type 3 IL-10 is ligated into the expression vector pET-21d(+), and then transformed into Rosetta(DE3) competent cells. After IPTG induction and centrifugation, the collected cells are disrupted using a cryogenic autoclave for 8–12 min of continuous cell disruption. Inclusion bodies are extracted and refolded at 4°C for 36 h using the dilution-renaturation method. The refolding solution is concentrated and replaced with protein buffer, then centrifuged at high speed (4°C, 10000×g, 5 min). The supernatant is purified using an AKTA protein purifier and then analyzed and verified by SDS-PAGE to obtain the recombinant protein of cyprinid herpesvirus type 3 IL-10. Then, mice were immunized with the obtained recombinant protein of cyprinid herpesvirus type 3 IL-10. Spleen cells were then fused with sp2 / 0 cells, and several hybridoma cell lines that secreted monoclonal antibodies against cyprinid herpesvirus type 3 IL-10 were screened. The antibody typing results all belonged to the IgG subclass (IgG1, IgG2a, and IgG2b). The screened hybridoma cell lines were purified and labeled to prepare monoclonal antibodies, which were then verified and identified using laser confocal microscopy and Western blotting techniques. The obtained monoclonal antibodies showed high specificity and strong stability and can be used in ELISA to detect the concentration of cyprinid herpesvirus type 3 IL-10 in carp blood and body fluids to predict the health status of fish, providing a detection method for monitoring the incidence of cyprinid herpesvirus type 3 in fish.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] 1. This invention obtains recombinant IL-10 protein of cyprinid herpesvirus type 3 through prokaryotic expression in Escherichia coli. After immunizing mice with the purified protein as an antigen, spleen cells are fused with sp2 / 0 cells, and hybridoma cell lines that can secrete monoclonal antibodies against cyprinid herpesvirus type 3 IL-10 are screened. These cells have the function of specifically recognizing cyprinid herpesvirus type 3, as well as good passage ability and can be amplified and cultured in large quantities, and can stably secrete monoclonal antibodies in vitro.

[0018] 2. The monoclonal antibody against Cyhvirus Type 3 IL-10 of the present invention can recognize the prokaryotic expression product, eukaryotic expression product, and extracellular expression functional region protein of Cyhvirus Type 3 interleukin-10. It can be applied to the immunological detection of CyHV-3-IL-10, including the specific detection of Cyhvirus Type 3 IL-10 concentration in carp blood or body fluid using ELISA technology, and the detection of Cyhvirus Type 3 IL-10 production in tissues using laser confocal microscopy through frozen tissue sections. It provides parameter indicators for the diagnosis, treatment, or prevention of Cyhvirus Type 3 infection diseases and has potential application value in fish disease prevention and control. Attached Figure Description

[0019] Figure 1 This is a diagram showing the purification and SDS-PAGE protein electrophoresis verification of the CyHv-3-IL-10 recombinant protein in Example 1.

[0020] Figure 2 The graph shows the antibody immunotiter detection results in Example 2. In the graph, 1 represents the blank and 2 represents the negative control.

[0021] Figure 3 In Example 2, the CyHV-3-IL-10 recombinant protein specifically reacted with the prepared monoclonal antibodies 1#, 3#, and 28#, showed a weaker specific reaction with 32#, and did not show a specific reaction with 14# and 26#.

[0022] Figure 4 The values ​​represent the antibody purity and antibody affinity constant in Example 2.

[0023] Figure 5 This is for the Western blot detection of monoclonal antibody specificity in Example 3.

[0024] Figure 6 Immunofluorescence analysis of the purified monoclonal antibody CyHV3-IL10 labeled with FITC; (A): transfection with pcDNA3.4 empty vector plasmid; (B): transfection with CyHV-3-IL-10 plasmid. Detailed Implementation

[0025] The present invention will be further described in detail below through specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, unless otherwise specified, the specific technical operations are all conventional operating procedures or conditions for those skilled in the art. Reagents or instruments used, unless otherwise specified, are all commercially available products.

[0026] The following examples will help those skilled in the art to further understand the present invention, but do not limit the invention in any way.

[0027] Example 1: Preparation of recombinant IL-10 protein of cyprinid herpesvirus type 3

[0028] (1) Induction of recombinant protein expression and inclusion body extraction

[0029] The coding region sequence of the cyprinid herpesvirus type 3 IL-10 gene was obtained from the NCBI database (accession number: NC_009127.1), and ligated into the expression vector pET-21d(+) to construct the pET-21d(+)-CyHV-3-IL-10 recombinant plasmid. This plasmid was then transformed into Rosetta(DE3) competent cells, and positive single colonies were picked and cultured until the bacterial culture reached OD. 600 When the pH value was between 0.6 and 0.8, IPTG (isopropyl thio-β-D-galactopyranoside, 1 mM) was used to induce expression. After large-scale induction of recombinant bacteria for 10 h, the cells were collected by centrifugation (7500 rpm, 5 min), and the cells were resuspended in sterile PBS. The cells were then disrupted using a continuous cell disruptor at ultra-low temperature and high pressure. After centrifugation, the supernatant was discarded, and the inclusion bodies were washed with Washing Buffer containing 1‰ DTT (dithiothreitol). After discarding the supernatant, the inclusion bodies were resuspended in Resuspension Buffer containing 1‰ DTT into a new, pre-weighed centrifuge tube. 20 μL of this resuspension was used for SDS-PAGE to determine the purity of the inclusion bodies. After centrifugation and discarding the supernatant, the inclusion bodies were dissolved in Dissolution Buffer containing 1% DTT to a final concentration of 30 mg / mL. The tubes were then stored at -20°C for later use.

[0030] (2) Refolding and purification of recombinant proteins

[0031] Prokaryotic proteins expressed in inclusion body form were refolded using a dilution-refolding method. After preparing the refolding solution and protein buffer, the mixture was filtered through a 0.22 μm nitrocellulose membrane and placed in a clean beaker. 10 mL of the inclusion body solution was added dropwise to 1 L of the prepared refolding solution, and in vitro refolding was performed with the aid of a magnetic stirrer (200 rpm) (36 h at 4°C). The diluted protein refolding solution was concentrated using a concentration cup and tube until the desired loading volume of 3–5 mL was reached (filtered through a 0.22 μm membrane before loading). Finally, the protein was purified by gel filtration chromatography using an AKTA protein purifier and a Superdex 200 column (GE Healthcare). Based on the peak positions of the purified proteins, protein peaks corresponding to their molecular weights were selected for concentration and SDS-PAGE analysis. The results showed that the purified CyHv-3-IL-10 protein exhibited single bands that matched the theoretical molecular weight (18 kDa). Figure 1 ).

[0032] Example 2: Preparation of monoclonal antibody against cyprinid herpesvirus type 3 IL-10

[0033] (1) Mouse immunization

[0034] The purified CyHV-3-IL-10 recombinant protein was provided to Beijing BGI Protein Research and Development Co., Ltd. for the preparation of monoclonal antibodies. The specific steps are as follows: BALB / c female mice were immunized with the purified CyHV-3-IL-10 recombinant protein at a dose of 30 to 60 μg each time, with an interval of two weeks, via intraperitoneal injection. After four immunizations, blood was collected from the orbital sinus, and the serum antibody titer was measured using enzyme-linked immunosorbent assay (ELISA). Once a certain value was reached, spleen cells from immunized mouse No. 1 (CyHV3-IL10-1) were collected for fusion. Figure 2 ).

[0035] (2) Cell fusion and screening

[0036] 1) Gently pipette healthy sp2 / 0 cells off the culture flask wall and transfer them into a 50mL centrifuge tube.

[0037] 2) The mice were euthanized by removing their eyeballs and drawing blood, then their necks were pulled and they were soaked in 75% alcohol for 5 minutes.

[0038] 3) Pour a small amount of serum-free IMDM into a petri dish, place the cell sieve and syringe core into the petri dish, remove the spleen of the mouse with scissors and tweezers, place it on the cell sieve, and gently crush the spleen thoroughly with the syringe core. Aspirate the crushed cells into a centrifuge tube containing sp2 / 0, and centrifuge at 1500 rad / min for 5 min.

[0039] 4) Remove the mouse's thymus with scissors and tweezers, crush it, put the crushed thymus cells into a 15mL centrifuge tube, add 2mL of HAT and 1mL of HT and place it in an incubator for later use.

[0040] 5) After centrifuging, discard the supernatant, carefully and gently reconstitute the cells with serum-free IMDM, and centrifuge (1500 rad / min, 5 min).

[0041] 6) Discard the supernatant of the centrifuged cells as much as possible, tap the bottom of the centrifuge tube to fully suspend the cells, place the centrifuge tube in 37°C warm water, slowly add 1 mL of PEG over about 1 minute, and let it stand in warm water for 1 minute after adding the PEG. Then slowly add 2 mL of serum-free IMDM over 2 minutes, followed by 8 mL of serum-free IMDM over 2 minutes. Centrifuge at 1000 rad / min for 5 minutes.

[0042] 7) Discard the supernatant, add 10 mL of serum, carefully blow the cells evenly, and pour them into the prepared thymocytes; then add sterile semi-solid culture medium to bring the volume to 50 mL and mix thoroughly; then pour evenly into 30 cell culture dishes, place the cell culture dishes in a humidified box, and then place them in an incubator for culture.

[0043] (3) Screening and subtype identification of monoclonal antibodies

[0044] After fusion, the cells were transferred to a semi-solid culture medium for culture. Single clones grown on the semi-solid medium were then transferred to 96-well plates for further culture and selection. The single clones in the 96-well plates were treated with cell culture medium for the first selection. Then, the selected clones were coated with CyHV-3-IL-10 and screened twice using ELISA to identify positive hybridoma cell lines. The resulting positive cell lines were subtype identified, cultured in large quantities, and cryopreserved. The specific ELISA procedure was as follows: the coating antibody was diluted to 0.5 μg / mL with 100 mM PBS buffer (pH 7.4), and 0.1 mL was added to each well. After incubation overnight at 4°C, the cells were washed three times with PBS-T. 200 μL of blocking buffer was added to each well, and the cells were incubated at 37°C for 2 h. After washing three times with PBS-T, 100 μL of hybridoma cell supernatant was added to each well, and the cells were incubated at 37°C for 1 h. After washing three times with PBS-T, add 0.1 mL of HRP-labeled antibody (SBA Clonotyping System-HRP, Southern Biotech) diluted 1:2000 with blocking buffer to each well and incubate at 37°C for 1 h. Wash three times with PBS-T; add 50 μL of substrate solution to each well and measure the absorbance at dual wavelengths (450, 630) within 10–20 min.

[0045] The screened positive strains were subjected to antibody subclass identification and Western blotting analysis (Table 1). CyhV-3-IL-10-1, cell #3 was IgG1 subtype, cells #14 and #16 were IgG2a subtype, and cells #28 and #32 were IgG2b subtype. Using the corresponding prokaryotic recombinant protein as the antigen, positive cell supernatant was used as the primary antibody (v / v = 1:4), and HRP-goat anti-mouse IgG as the secondary antibody (v / v = 1:5000). Several monoclonal antibody cell supernatants with good specificity were ultimately screened from CyHV-3-IL-10. Specifically, the CyHV-3-IL-10 recombinant protein showed a specific reaction with cell lines #1, #3, and #28 of the monoclonal antibody cell supernatant used to immunize mice; cell line #32 showed a weaker specific reaction; and cells #14 and #26 showed no specific reaction. Figure 3 ).

[0046] Table 1: Identification of Monoclonal Cell Line Subclasses and Related Information

[0047]

[0048] (4) Purification and labeling of monoclonal antibodies

[0049] From the positive monoclonal clones selected from CyHV-3-IL-10, cell line 1 (CyHV31-IL10) antibody was selected for purification and labeling. The monoclonal antibody was purified by affinity chromatography using a HiTraprProtein A FF column. Antibody purity was detected by SDS-PAGE, and the antibody affinity constant was determined by ELISA (affinity constant = 150000 × A / antibody concentration, where A represents the antibody dilution factor corresponding to half of the maximum OD value; OD value was measured using dual wavelengths at 450 nm and 630 nm). The CyHV31-IL10 antibody was selected for purification and named CyHV3-IL-10-1. The monoclonal antibody was then labeled with FITC (fluorescein isothiocyanate). Antibody purification and labeling were performed by Beijing BGI Protein Research Center Co., Ltd., and the affinity constant of the monoclonal antibody was 2.74E+09. Figure 4 ).

[0050] Example 3: Identification and Specificity Analysis of Monoclonal Antibodies Against Cyprinid Herpesvirus Type 3 IL-10

[0051] (1) Western blot antibody specificity detection

[0052] Recombinant eukaryotic protein of cyprinid herpesvirus type 3 IL-10 was subjected to SDS-PAGE gel electrophoresis at 150V for 50 min. After electrophoresis, protein gel transfer was performed using a protein transfer instrument (25V, 2.5A, 7 min). After transfer, the membrane was blocked with 5% skim milk blocking buffer for 1 h. Then, an appropriate amount of 1:1000 diluted primary antibody (serum) was placed in an antibody incubation box and incubated at room temperature for 2 h or overnight at 4°C. The PVDF membrane was washed three times with TBST for 5 min each time, and then an appropriate amount of HRP-labeled goat anti-mouse secondary antibody diluted 1:10000 was added. The membrane was incubated at room temperature for 1 h, and then washed three times with TBST for 5 min each time. Finally, the membrane was placed in an Odyssey CLx near-infrared fluorescence imaging system for scanning and imaging. Figure 5 The bands produced by CyHV-3-IL-10 incubation were slightly larger than the theoretical molecular weight, but the bands were single. It is speculated that the eukaryotic protein CyHV-3-IL-10 may undergo chemical modifications such as glycosylation and phosphorylation during expression, which may have led to the phenomenon that the protein bands are larger than the theoretical molecular weight.

[0053] (2) Direct immunofluorescence detection analysis

[0054] HEK293T cells were cultured in 6-well plates with cell spreaders. When the cell confluence reached approximately 90%, they were transfected with the pcDNA3.4-CyHV-3-IL-10-His plasmid. Cells transfected with the empty pcDNA3.4-His plasmid served as a control group. 24 hours after transfection, the culture supernatant was discarded, and the cells were washed with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, removed, and washed three times with PBS buffer for 5 minutes each time. The cells were then permeated with 0.5% Triton-X100 at room temperature for 10 minutes, and a sufficient amount of blocking buffer (5% BSA) was added. The cells were incubated at room temperature for 1 hour, and the blocking buffer was discarded. The control and experimental groups were then incubated with FITC-labeled CyHV-3-IL-10-1-FITC monoclonal antibody diluted 1:200 with 5% BSA at room temperature for 2 hours. Wash three times with PBS buffer, then add DAPI for staining at room temperature for 10 min, wash three times with PBS buffer for 5 min each time, add anti-fluorescence quencher, mount, and observe and photograph under a laser confocal microscope. Results showed that CyHV-3-IL-10-1 could recognize CyHV-3-IL-10 protein located in the cytoplasm of HEK293T cells, while no specific fluorescent signal or positive cells were observed in the control group. This indicates that the prepared CyHV-3-IL-10-1 monoclonal antibody can specifically bind to CyHV-3-IL-10 protein. Figure 6 ).

[0055] The recombinant protein of cyprinid herpesvirus type 3 IL-10 and the monoclonal antibody against cyprinid herpesvirus type 3 IL-10 obtained through the above embodiments have the following application prospects:

[0056] Recombinant protein of cyprinid herpesvirus type 3 IL-10 can serve as a potential molecular adjuvant. Monoclonal antibodies against cyprinid herpesvirus type 3 IL-10 can be used for in vitro ELISA detection of cyprinid herpesvirus type 3 IL-10 concentration and for the preparation of kits for the immunodetection of cyprinid herpesvirus type 3.

[0057] Specifically, the ELISA method for detecting the secretion level of IL-10 protein of cyprinid herpesvirus type 3 is as follows:

[0058] (1) Coating: Dilute the prepared cyprinid herpesvirus type 3 IL-10 monoclonal antibody with coating buffer to a protein content of 1–10 μg / mL, add 100 μL to each reaction well of the ELISA plate, and incubate at 37°C for 2 h or 4°C overnight.

[0059] (2) Washing: Discard the liquid in the wells, spin dry, and wash the plate 3 times with washing buffer for 3 minutes each time (hereinafter referred to as washing). Spin dry (you can also gently tap to dry the liquid in the wells).

[0060] (3) Blocking: Use 10mM PBST (10mM PBS, 0.05% Tween-20) containing 1% BSA or 5% skim milk as the blocking solution, 350–400 μl / well, 37℃, 2h.

[0061] (4) Washing the plate: Same as step (2).

[0062] (5) Sample addition: Add 100 μL of the diluted sample to the above-mentioned coated reaction wells and incubate at 37°C for 1 hour. Then wash (set up blank wells, negative control wells, positive control wells, standard wells, and sample wells respectively).

[0063] (6) Add the primary antibody to detect IL-10 antibody. Add 100 μL to each well of the ELISA plate and incubate at 37°C for 2 hours.

[0064] (7) Add enzyme-labeled antibody: Add 100 μL of freshly diluted enzyme-labeled antibody (after titration) to each reaction well, incubate at 37°C for 0.5–1 hour, and wash.

[0065] (8) Add substrate solution for color development: Add 100 μL of the temporarily prepared TMB substrate solution to each reaction well and incubate at 37°C for 10–30 minutes.

[0066] (9) Termination of reaction: Add 50 μL of 2M sulfuric acid to each reaction well.

[0067] (10) Measure the OD of each well using an ELISA analyzer. 450 value.

[0068] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A hybridoma cell strain secreting anti-CyHV3 IL-10 monoclonal antibody, named CyHV31-IL10, which has been deposited with the China Center for Type Culture Collection on August 17, 2022, and has the accession number CCTCC C2022212.

2. An anti-CyHV3 IL-10 monoclonal antibody, which is obtained by the hybridoma cell strain secreting anti-CyHV3 IL-10 monoclonal antibody according to claim 1.

3. Use of the anti-CyHV3 IL-10 monoclonal antibody according to claim 2 in the preparation of a reagent for diagnosing or detecting CyHV3 infection.

4. A kit for immunologically detecting CyHV3, comprising the anti-CyHV3 IL-10 monoclonal antibody according to claim 2.

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