Monoclonal antibody for resisting Marek's disease virus US3 protein and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an anti-Marek's disease virus US3 protein monoclonal antibody and application thereof. The invention provides an anti-Marek's disease virus US3 protein monoclonal antibody which is secreted by a hybridoma cell with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.46324. The antibody is strong in specificity and high in sensitivity, can be used for directly detecting the MDV US3 protein by an immunofluorescence method, realizes rapid diagnosis of MDV infection, and provides technical support for further researching the biological function of the MDV US3 protein and further revealing an MDV pathogenic mechanism.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to an anti-Marek's disease virus US3 protein monoclonal antibody and application thereof. BACKGROUND

[0002] Marek's disease (MD) is a lymphoproliferative neoplastic disease caused by Marek's disease virus (MDV) and is highly contagious in chicken flocks. Since the late 1960s, MD has been effectively controlled through vaccination, but MDV has been continuously adapting and mutating, breaking through the protection of existing vaccines, especially the emergence of very virulent plus MDV (vv+MDV) with stronger neurotoxicity and immunosuppressive properties, resulting in the continued occurrence of MD in the global poultry industry. In addition to the increase in MDV virulence, mature virions have strong resistance to the environment and can persist in the environment, threatening the health of chicken flocks. The diagnosis of MD can be initially determined through clinical features such as animal cleft, iris discoloration, and skin nodules, but these clinical symptoms often appear mixed and cannot be directly determined, so it mainly relies on pathogen isolation, antigen-antibody detection, polymerase chain reaction (PCR), and other methods for identification.

[0003] MDV belongs to the Marek's disease virus genus of the alpha herpesvirus subfamily of the herpesvirus family. The MDV genome is composed of unique long (UL) and short (US) regions, with inverted terminal and internal repeat long (TRL, IRL) and short (TRS, IRS) regions on both sides of each region. The US3 gene encodes a serine / threonine protein kinase in the US region of the viral genome and is relatively conserved in alpha-herpesviruses, which has a regulatory role in virus replication. Studies have shown that the US3 protein plays an important role in virus replication, immune escape, inhibition of apoptosis, inhibition of protein translation, and decomposition of the cytoskeleton during MDV infection.

[0004] MDV US3 protein can destroy the structure of promyelocytic leukemia protein (PML) and Sp100 protein by phosphorylation modification, promote virus replication. It can also interact with IRF7 and the subunit p65 and p50 of NF-κB, inhibit the production of IFN-β and interleukin 6 (IL-6), escape the host innate immune response. The study of Liao et al. showed that MDV US3 can phosphorylate chicken histone deacetylase 1 (chHDAC1) and chicken histone deacetylase 2 (chHDAC2), promote the expression of cell cycle regulatory factors p21 and p27, and inhibit apoptosis. The study of Wang et al. found that MDV US3 interacts with Wilms tumor factor 1 associated protein (WTAP) in the methylation complex and is phosphorylated, reduces the level of cellular N6-methyladenosine and inhibits protein translation. The results of the study of Schumacher et al. showed that MDV US3 can block the binding of RhoA to Rho-associated kinase (ROCK), inhibit myosin light chain phosphorylation, reduce the stability and formation of stress fibers, promote the breakage of actin stress fibers, and promote the spread of the virus.

[0005] Currently, there is no related report on the anti-MDV US3 protein monoclonal antibody, and the function of MDV US3 protein is mostly studied by using exogenous expression protein, which limits the further exploration of its biological function, and the molecular mechanism of how the protein accurately regulates virus replication is still in the exploratory stage. The development of anti-MDV US3 protein monoclonal antibody which can be used for direct immunofluorescence method not only can realize the rapid diagnosis of MDV infection, but also will greatly promote the in-depth analysis of the biological function of MDV US3 protein and the pathogenic mechanism of MDV. SUMMARY

[0006] In order to realize the rapid diagnosis of MDV infection, the application provides an anti-MDV US3 protein monoclonal antibody, which is secreted by a hybridoma cell with a preservation number of CGMCC No. 46324.

[0007] A hybridoma cell strain secreting the anti-MDV US3 protein monoclonal antibody is also provided, and the preservation number thereof is CGMCC No. 46324.

[0008] A kit for detecting MDV US3 protein is also provided, which comprises the anti-MDV US3 protein monoclonal antibody.

[0009] The kit can also comprise general reagents for direct immunofluorescence method. The general reagents comprise fluorescent substances for binding the anti-MDV US3 protein monoclonal antibody. The fluorescent substances can be fluorescein isothiocyanate (FITC).

[0010] The kit can also comprise general reagents for indirect enzyme-linked immunosorbent assay or indirect immunofluorescence method. The general reagents include signal marker labeled anti-mouse IgG antibody for binding the anti-MDV US3 protein monoclonal antibody. The signal marker can be horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC).

[0011] Also provided is a method for preparing the anti-MDV US3 protein monoclonal antibody, which comprises injecting the hybridoma cell strain with the preservation number of CGMCC No. 46324 into the abdominal cavity of a mouse, collecting ascites, and purifying the antibody from the ascites.

[0012] The use of the anti-MDV US3 protein monoclonal antibody in the preparation of a product for detecting MDV US3 protein also belongs to the scope of the present application. The product can be a reagent or a kit for detecting MDV US3 protein by direct immunofluorescence method, indirect enzyme-linked immunosorbent assay or indirect immunofluorescence method.

[0013] The present application obtains 6His-MDV US3 protein by constructing recombinant expression plasmid pFastBac1-US3 of MDV US3 gene, transforming into E. coli DH10Bac competent cells, picking white single colonies for expansion culture after blue-white spot screening, extracting recombinant bacmid rBacmid-US3, and using an insect baculovirus expression system for expression. Compared with other expression systems, the insect baculovirus expression system can efficiently express foreign genes in the late stage of virus infection through polyhedron protein promoter, and can perform glycosylation, phosphorylation and other modifications on the expression product relying on the endogenous post-translational modification system in eukaryotic cells, so that the 6His-MDV US3 protein can be closer to the structure and function of the natural protein, and has good antigenicity and biological function.

[0014] The 6His-MDV US3 protein is used to immunize mice, and the spleen is taken out for cell fusion. The positive hybridoma cells capable of stably secreting antibodies are screened out by indirect ELISA method and indirect immunofluorescence method after 4 times of subcloning, and are named MDV-US3. The paraffin oil is injected into the abdominal cavity of the mouse, and the positive hybridoma cells are injected into the abdominal cavity of the mouse after expansion culture to prepare ascites, and the anti-MDV US3 protein monoclonal antibody is obtained after purification. The antibody is IgG2a subclass, the light chain is Kappa type, has good reactivity and affinity, and the Kd value is 4.34nM ( Figure 6 ). The specific detection result shows that the antibody can specifically bind to MDV US3 protein, and has no cross reaction with Ch-IFNγ, Ch-IL-2, MDV pp38, MDV gB and MDV Meq protein antigens ( Figure 10Sensitivity assays showed that the anti-MDV US3 protein monoclonal antibody at a concentration of 0.03125 μg / mL could still detect MDV US3 protein, with a sensitivity far exceeding that of the anti-MDV US3 protein polyclonal antibody. Figure 11 ).

[0015] A direct immunofluorescence method for detecting MDV US3 protein was established using FITC-labeled monoclonal antibodies against MDV US3 protein. Compared with commonly used indirect immunofluorescence methods and other conventional diagnostic techniques, the direct immunofluorescence method of this invention simplifies the operation process and shortens the detection time while maintaining high specificity, providing a new visual detection strategy for rapid laboratory diagnosis of MDV infection. Experiments have demonstrated that the detection effect of the direct immunofluorescence method of this invention (…) Figure 12 A) Detection efficacy compared to indirect immunofluorescence assay ( Figure 12 C) Similar to MDV, but with a shorter detection time, it can more specifically, quickly, and visually detect the cellular localization of MDV US3 protein, enabling rapid diagnosis of MDV infection and providing technical support for further research on the biological function of MDV US3 protein and thus revealing the pathogenesis mechanism of MDV.

[0016] The biomaterial preservation information of this invention is as follows:

[0017] Biomaterial: MDV-US3

[0018] Classification and nomenclature: Hybridoma cell lines

[0019] Deposit date: January 16, 2025

[0020] Accession number: CGMCC No. 46324

[0021] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0022] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Attached Figure Description

[0023] Figure 1 PCR identification results of recombinant rod-like particle rBacmid-US3; M: DNA molecular weight standard; 1: Amplification products using rBacmid-US3 as template and primers US3-F and US3-R; 2: Amplification products using rBacmid-US3 as template and primers M13-F and M13-R.

[0024] Figure 2The pathological changes of the cells (A) and the results of indirect immunofluorescence identification (B) after the insect ovary cells (SF9) were transfected with the recombinant bacmid rBacmid-US3; Mock represents the control group; 48h, 72h, 96h represent the time (hours) after the SF9 cells were transfected with the recombinant bacmid rBacmid-US3.

[0025] Figure 3 The results of Western blotting identification of the small amount of 6His-MDV US3 protein expressed by the Spodoptera exigua ovary cells (HF5); 48h, 60h, 72h, 84h, 96h, 108h, 120h represent the time (hours) after the third generation of recombinant baculovirus liquid (rBaculovirus-US3-P3) was inoculated into the HF5 cells; ku represents kilodaltons.

[0026] Figure 4 The results of SDS-PAGE (left) and Western blotting identification (right) of the 6His-MDV US3 protein; M, protein molecular weight marker; 1, the supernatant of the HF5 cell culture; 2, the purified 6His-MDV US3 protein.

[0027] Figure 5 The SDS-PAGE gel map of the purified anti-MDV US3 protein monoclonal antibody; ku represents kilodaltons.

[0028] Figure 6 The affinity identification results of the anti-MDV US3 protein monoclonal antibody.

[0029] Figure 7 The results of Western blotting identification of the monoclonal antibody; M, protein molecular weight marker; 1, the results of Western blotting identification of the 6His-MDV US3 protein using the anti-MDV US3 protein monoclonal antibody as the primary antibody; 2, the results of Western blotting identification of the 6His-MDV US3 protein using the anti-6His tag monoclonal antibody as the primary antibody.

[0030] Figure 8 The results of indirect immunofluorescence (IFA) identification of the reaction of the anti-MDV US3 protein monoclonal antibody with the US3 protein in the chicken embryo fibroblast cells (CEF cells) infected with the MDV / RB1B strain; A, negative control; B, the results of IFA identification using the anti-MDV US3 protein polyclonal antibody as the primary antibody; C, the results of IFA identification using the anti-MDV US3 protein monoclonal antibody as the primary antibody.

[0031] Figure 9The results of indirect immunofluorescence (IFA) identification of the reactivity of the anti-MDV US3 protein monoclonal antibody with the HA-US3 protein in CEF cells transfected with pcDNA3.1-HA-US3 plasmid; A, negative control; B, IFA identification results using the anti-MDV US3 protein monoclonal antibody as the primary antibody; C, IFA identification results using the anti-hemagglutinin (HA) tag antibody as the primary antibody.

[0032] Figure 10 The results of specific detection of the anti-MDV US3 protein monoclonal antibody.

[0033] Figure 11 The results of sensitivity detection of the anti-MDV US3 protein monoclonal antibody; the columnar chart uses unpaired t test to analyze the significance of the difference between the two groups of data, P<0.05 indicates that there is a difference between the two groups of data, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0034] Figure 12 The results of MDV immunofluorescence detection; A, the results of MDV detection by direct immunofluorescence using the anti-MDV US3 protein monoclonal antibody; B, the negative control group of direct immunofluorescence; C, the results of MDV detection by indirect immunofluorescence using the anti-MDV US3 protein monoclonal antibody; D, the negative control group of indirect immunofluorescence. DETAILED DESCRIPTION

[0035] The application will be described in detail below in conjunction with examples, and it should be understood that the following examples are only used to explain and describe the application, and do not limit the scope of the application in any way.

[0036] The biological materials used in the following examples are as follows:

[0037] Insect ovary cells (spodoptera frugiperda clone 9, SF9), heliothis armigera ovary cells (BTITN5 B1-4, HF5), mouse myeloma cells (SP2 / 0), E. coli DH10Bac competent cells, and chicken embryo fibroblasts (Chicken embryo fibroblast, CEF) were provided by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, and can also be purchased through commercial channels.

[0038] The MDV / RB1B strain is described in the literature "Jin, Huan; Kong, Zimeng; Mehboob, Arslan; Jiang, Bo; Xu, Jian; Cai, Yunhong; Liu, Wenxiao; Hong, Jiabing; Li, Yongqing*Transcriptional Profiles Associated with Marek's Disease Virus in Bursa and Spleen Lymphocytes Reveal Contrasting Immune Responses during Early Cytolytic Infection. Viruses, 2020;12(3):354." The strain is available to the public from the Institute of Animal Science and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, for use in repeating the present application only.

[0039] Chicken interferon gamma (Ch-IFNγ) represented by SEQ ID NO: 4, chicken interleukin 2 (Ch-IL-2) represented by SEQ ID NO: 5, Marek's disease virus phosphorylated protein 38 (MDV pp38) represented by SEQ ID NO: 6, Marek's disease virus gB protein (MDV gB) represented by SEQ ID NO: 7, and Marek's disease virus Meq protein (MDV Meq) represented by SEQ ID NO: 8 were prepared by the Institute of Animal Science and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences.

[0040] Anti-MDV serum is an anti-Marek's disease virus serum obtained by immunizing chickens with a Marek's disease live vaccine (CVI988 / Rispens strain) and prepared by the Institute of Animal Science and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences.

[0041] The pcDNA3.1-HA-US3 plasmid is a recombinant expression vector obtained by inserting the HA-US3 gene represented by SEQ ID NO: 3 into the Xhol site and the BamHl site of the pcDNA3.1 plasmid and prepared by the Institute of Animal Science and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences. The pcDNA3.1 plasmid can be purchased through commercial channels.

[0042] The pFastBac1 is an expression vector for high-level expression of proteins in insect cells and provided by Beijing Genkey Biotechnology Co., Ltd.

[0043] 6-8-week-old healthy female BALB / c mice were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0044] The main reagents used in the following examples are as follows:

[0045] DMEM medium and fetal bovine serum (FBS) were purchased from Gibco. Freund's complete adjuvant, Freund's incomplete adjuvant, HAT Supplement (50x), HT Supplement (100x), fusion agent PEG1450, mouse anti-hemagglutinin (HA) tag antibody, goat anti-mouse-IgG-HRP were purchased from Sigma. HiPure Plasmid Miniprep Kit, Mouse Rapid Isotyping Kit, Goat Anti-Mouse-IgG-FITC, IgG Elution Buffer and Protein A / G IgG Binding Buffer were purchased from Thermo Fisher. DNA extraction kit was purchased from QIAGEN. 4% tissue cell fixative, ELISA termination solution, TMB single-component color developing solution, triton X-100 were purchased from Solarbio. Anti-6His tag monoclonal antibody was purchased from ABclonal. Fluorescein 5-isothiocyanate was purchased from Zibo Yunhui Biological Chemical Technology Co., Ltd.

[0046] Unless otherwise specified, the reagents used in the following examples are all conventional reagents in the art, which can be commercially available or prepared according to conventional methods in the art. Unless otherwise specified, the experimental methods and conditions used in the following examples are all conventional experimental methods and conditions in the art, which can be referred to relevant experimental manuals, known literatures or manufacturer's instructions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0047] Example 1. Obtaining of anti-Marek's disease virus US3 protein monoclonal antibody

[0048] 1. Construction and identification of recombinant bacmids

[0049] The MDV US3 gene shown in SEQ ID NO: 1 was synthesized by Beijing Nankai Biotechnology Co., Ltd. and PCR primers thereof were synthesized. The MDV US3 gene was ligated to a pFastBac1 vector to obtain a recombinant plasmid pFastBac1-US3. 100 μL of E. coli DH10Bac competent cells were placed on ice for 5 min. 2 μL of the pFastBac1-US3 plasmid was added, and the tube was mixed gently with fingers, and then was placed in an ice bath for 30 min. The EP tube was placed in a 42°C water bath for 45 seconds. After the timer went off, the tube was inserted back into the ice for 2 min. In a clean bench, 900 μL of LB liquid medium was added to the tube, and the tube was cultured at 37°C for 4 hours at 225 r / min. 40 μL of X-Gal was applied to a three-antibiotic plate (LB solid medium containing 50 μg / mL of kanamycin, 7 μg / mL of gentamicin, 10 μg / mL of tetracycline and 40 μg / mL of IPTG) 1 hour in advance. 100 μL and 500 μL of the cultured bacterial liquid were applied to two three-antibiotic plates, respectively. The plates were placed in a 37°C oven for two days. After a white single colony was picked and expanded, a HiPure plasmid extraction kit (Thermo Fisher, K210007) was used to extract the recombinant bacmid according to the kit instructions. PCR identification was performed using universal primers (M13-F and M13-R) and US3 gene upper and lower primers (US3-F and US3-R), and the PCR product was sent to Beijing Nankai Biotechnology Co., Ltd. for sequencing. The nucleotide sequences of the primers are as follows:

[0050] US3-F: 5'-ATGTCTTCGAGTCCGGAGGCAG-3'

[0051] US3-R: 5'-AGCGGCAGTTATCGTGTATAATG-3'

[0052] M13-F: 5'-GTTTTCCCAGTCACGAC-3'

[0053] M13-R: 5'-CAGGAAACAGCTATGAC-3'

[0054] The results are shown in Figure 1 . The US3-F and US3-R amplification product bands were about 1000 bp, and the M13-F and M13-R amplification product bands were about 3000 bp, which were consistent with the expected amplification fragment size and the sequencing results were correct, proving that the recombinant bacmid was successfully constructed. The recombinant bacmid with correct sequencing results was named rBacmid-US3.

[0055] 2. Rescue of recombinant baculovirus

[0056] The recombinant bacmid rBacmid-US3 was transfected into insect ovary cells (spodoptera frugiperda clone 9, SF9) for culture, and microscopic observation was performed at 48, 72, and 96 hours (h) after transfection; at the same time, indirect immunofluorescence (IFA) identification was performed using anti-6His tag monoclonal antibody (ABclonal, AE003) as a primary antibody and goat anti-mouse-IgG-FITC (Thermo Fisher, A11001) as a secondary antibody; and normal cultured SF9 cells were used as a control group (Mock). After successful transfection, centrifugation was performed at 8000 r / min for 5 min, and the cell culture supernatant was collected to obtain the first generation of recombinant baculovirus liquid rBaculovirus-US3-P1 expressing 6His-MDV US3 protein. The SF9 cells were infected with rBaculovirus-US3-P1, and after 96 h of suspension culture at 27°C and 115 r / min, the cell morphology was observed, the cell culture supernatant was collected by centrifugation, and the second generation of recombinant baculovirus liquid rBaculovirus-US3-P2 was obtained. In the same way, the third generation of recombinant baculovirus liquid rBaculovirus-US3-P3 was obtained.

[0057] The results are shown in Figure 2 Compared with the control group (Mock), the SF9 cells infected with the recombinant bacmid rBacmid-US3 showed a slower growth rate, and the cell diameter increased after 72 h, with shrinkage and even death Figure 2 A). The IFA identification results showed obvious fluorescence, indicating the expression of 6His-MDV US3 protein Figure 2 B).

[0058] 3. Small-scale expression of recombinant MDV US3 protein and Western blotting identification

[0059] Take the appropriate amount of the third generation of recombinant baculovirus liquid rBaculovirus-US3-P3 infection of Spodoptera frugiperda ovary cells (BTITN5 B1-4, HF5), under the condition of 27℃, 115 r / min suspension culture; from 48 to 120 hours (hpi) after inoculation, every 12 h cell suspension was centrifuged at 4℃, 8000 r / min for 10 min, and 100 μL supernatant was taken for sample preparation; the samples taken at different time points were loaded in equal amounts for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the protein gel was transferred to PVDF membrane with 100V constant voltage; after 2h of transfer, the membrane was soaked in 5% skim milk at 4℃ overnight for blocking; the anti-6His tag monoclonal antibody (ABclonal, AE003) was diluted according to the ratio of 1:5000 and used as the primary antibody, and incubated at room temperature for 2h; after washing with PBST for 4 times, goat anti-mouse-IgG-HRP (Sigma, 12-349) was diluted according to the ratio of 1:10000 and used as the secondary antibody, and incubated at room temperature for 1h; after washing with PBST for 4 times, color development was performed with color developing solution and exposed to photography, and the protein expression was observed.

[0060] The results are shown in Figure 3 HF5 cells reached the highest expression of 6His-MDV US3 protein 96h after inoculation with rBaculovirus-US3-P3. The culture supernatant of HF5 cells 96h after inoculation with rBaculovirus-US3-P3 was selected for protein purification.

[0061] 4. Expression and purification of recombinant MDV US3 protein

[0062] Take an appropriate amount of the third generation of recombinant baculovirus liquid rBaculovirus-US3-P3 to infect HF5 cells, and culture under the condition of 27℃, 115 r / min suspension for 96h, and centrifuge at room temperature 9000 r / min for 5min. Collect the cell culture supernatant, mix 100 μL supernatant with 20 μL 5× protein loading buffer (containing DTT) and boil for 10 min for sample preparation, and perform SDS-PAGE identification to observe protein expression. After confirming protein expression, 6His-MDV US3 protein was purified by nickel column. The purified protein was identified by Western blotting (method same as above) and liquid chromatography-mass spectrometry analysis.

[0063] The results of SDS-PAGE and Western blotting identification showed that the purified 6His-MDV US3 protein was obtained Figure 4). The 6His-MDV US3 protein band size was higher than the predicted band size of the target gene, which might be because the target protein was modified by the eukaryotic endogenous post-translational modification system during protein expression. Liquid chromatography-mass spectrometry analysis results showed that the purified 6His-MDV US3 protein was indeed the target protein. The amino acid sequence of the MDV US3 protein is shown in SEQ ID NO: 2. The purified 6His-MDV US3 protein was expressed in large quantities, and the protein was concentrated by centrifugation at 4°C, 3500 r / min for 30 min using an ultrafiltration tube, and then replaced with pre-cooled PBS for 3 times, and the protein concentration was determined by BCA protein concentration detection method.

[0064] 5. Mouse immunization and detection of serum titer

[0065] 6-8 week-old healthy female BALB / c mice were immunized with the above purified 6His-MDV US3 protein as the immunogen at 100 μg per mouse. The mouse serum titer was detected by indirect ELISA method. The indirect ELISA method is as follows: dilute the 6His-MDV US3 protein to 1 μg / mL with coating solution, add 100 μL per well to a 96-well enzyme-labeled plate, and coat overnight at 4°C; add 5% skim milk, block at 37°C for 2 h; wash 5 times with PBST; dilute the negative serum and positive serum from 1:1000 to 1:128000 with 5% skim milk, and add the serum at each dilution to the enzyme-labeled plate as the primary antibody, 100 μL per well, and incubate at 37°C for 1 h; wash 5 times with PBST; dilute goat anti-mouse IgG-HRP (Sigma, 12-349) 1:1000 with 5% skim milk as the secondary antibody, add to the enzyme-labeled plate, 100 μL per well, and incubate at 37°C for 45 min; wash 5 times with PBST; add 100 μL TMB single-component color developing solution to each well, develop color at 37°C for 15 min; add 50 μL ELISA stop solution to each well, stop the reaction, and then place in an enzyme-labeled instrument to detect the OD450nm value; calculate the P / N value, P / N = positive serum OD450nm value / negative serum OD450nm value), and P / N ≥ 2.1 is determined as positive. The results are shown in Table 1, and the serum titer of mouse 23# is the highest.

[0066] Table 1: Serum titer (OD450nm) of 6His-MDV US3 protein immunized mice

[0067]

[0068] 6. Cell fusion and establishment of hybridoma cell strain

[0069] The mouse with the highest serum titer, 23#, was sacrificed after 3 days of booster immunization, and the spleen was removed to prepare a spleen cell suspension. 1×10 810 spleen cells and 2×10 7 After mixing mouse myeloma cells (SP2 / 0), PEG1450 fusion agent was slowly added to initiate cell fusion, followed by the slow addition of 25 mL of preheated DMEM medium to terminate the fusion. The cell suspension was centrifuged at 800 r / min for 7 min at room temperature, the supernatant was discarded, and the cells were resuspended in 65 mL of preheated HAT-containing medium. 100 μL of the fused cells were added to each well of a 96-well cell culture plate containing 100 μL of feeder cells, and the plates were incubated at 37°C in a 5% CO2 incubator. After 7–10 days, the hybridoma cell culture supernatant was analyzed using the aforementioned indirect ELISA and indirect immunofluorescence (IFA) methods to screen for positive wells.

[0070] Indirect immunofluorescence method: Chicken embryonic fibroblasts (CEF) were seeded at 80% cell density in 96-well cell culture plates. After 24 hours, 5 pfu of MDV / RB1B strain was added to each well. After culturing for 96 hours, the cells were fixed with 4% tissue cell fixative on ice for 15 minutes, followed by permeabilization with 0.1% Triton X-100 on ice for 15 minutes. The cells were washed twice with PBST and blocked overnight at 4°C with 5% BSA. 50 μL of anti-MDV serum diluted 1:1000 with 5% BSA was added to each well and incubated at room temperature for 2 hours. The cells were washed four times with PBST. 100 μL of hybridoma cell culture supernatant was added to each well and incubated overnight at 4°C. The cells were washed four times with PBST. Goat anti-mouse IgG-FITC (Thermo-21-21-3 ... Fisher (A11001) and goat anti-chicken-IgG-TRITC (ThermoFisher, A11039), 50 μL per well, incubated at room temperature for 1 h; washed 4 times with PBST; added 50 μL of DAPI staining solution diluted 1:1000 with PBS per well, incubated at room temperature for 2 min; washed 4 times with PBST, and observed and photographed using an inverted fluorescence microscope.

[0071] The cell wells that tested positive were subcloned using a limiting dilution method until positive wells containing only a single cell cluster that secreted antibodies were selected. These cell clusters were then expanded and cultured. After four subcloning cycles, one hybridoma cell line was selected and named MDV-US3. Hybridoma cell line MDV-US3 was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 46324 on January 16, 2025.

[0072] 7. Ascites preparation and antibody purification

[0073] 0.5 mL of paraffin oil was injected intraperitoneally into 6–8 week old female BALB / c mice. Seven days later, 1 × 10⁻⁶ g of paraffin oil was injected intraperitoneally into the mice.6 MDV-US3 hybridoma cells. After 7-10 days, the mice were observed, and if the mice had significant abdominal swelling, the ascites were extracted. The ascites were purified using the Thermo protein A / G purification system to obtain the anti-MDV US3 protein monoclonal antibody. SDS-PAGE was used to detect the antibody purification, and the results showed that there was a protein band at 55 ku and 25 ku (Fig. 3). Figure 5 The anti-MDV US3 protein monoclonal antibody was concentrated using an ultrafiltration tube.

[0074] 8. Affinity determination of monoclonal antibody

[0075] The affinity of the anti-MDV US3 protein monoclonal antibody was determined by indirect ELISA. The method is as follows: 5 μg / mL and 2.5 μg / mL 6His-MDV US3 protein were used to coat the enzyme-labeled plate, 100 μL per well, 4 ℃ coating overnight; washed 5 times with PBST; added 5% skim milk, 200 μL per well, 37 ℃ blocked for 2 h; washed 5 times with PBST; added anti-MDV US3 protein monoclonal antibody with concentrations of 18, 9, 4.5, 2.25, 1.125, 0.563, 0.281 and 0.141 μg / mL, respectively, 100 μL per well, 37 ℃ incubated for 1 h; washed 5 times with PBST; added goat anti-mouse IgG-HRP (Sigma, 12-349) diluted 1:1000 with 5% skim milk, 100 μL per well, 37 ℃ incubated for 45 min; washed 5 times with PBST; added 100 μL TMB single-component color developing liquid to each well, 37 ℃ reacted for 15 min in the dark; added 50 μL ELISA stop solution to each well, and after the reaction was stopped, the OD450nm value was determined (Fig. 4). Figure 6 The affinity constant (Kd) was calculated according to the method described in the literature "Cao MY, Wang J, Zhou LY, et al. Preparation of monoclonal antibody against chicken promyelocytic leukemia protein and its application in fluorescence detection [J]. Chinese Journal of Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4500-4509." The results showed that the anti-MDV US3 protein monoclonal antibody had high affinity, Kd=4.34 nM.

[0076] 9. Class and subclass determination of monoclonal antibody

[0077] The class and subclass of the anti-MDV US3 protein monoclonal antibody were identified using the mouse rapid antibody isotype detection kit (Thermo Fisher, A38550) according to the kit instructions. The results showed that the anti-MDV US3 protein monoclonal antibody was of IgG2a subclass, and the light chain was of Kappa type.

[0078] 10. Western blotting identification of monoclonal antibody

[0079] The 6His-MDV US3 protein was sampled, and after SDS-PAGE, the protein gel was transferred to a PVDF membrane at a constant voltage of 100 V. After transfer, the PVDF membrane was immersed in 5% skim milk for 2 hours at room temperature. The prepared anti-MDV US3 protein monoclonal antibody was diluted at 1:1000, and the anti-6His tag monoclonal antibody (ABclonal, AE003) was diluted at 1:5000, and they were used as the primary antibody, respectively, and incubated at room temperature for 2 hours. After washing with PBST for 4 times, goat anti-mouse IgG-HRP (Sigma, 12-349) was diluted at 1:10000 and used as the secondary antibody, and incubated at room temperature for 1 hour. After washing with PBST for 4 times, color development was performed with color developing solution and exposed to photography. As shown in Figure 7 , the 6His-MDV US3 protein was detected with the anti-MDV US3 protein monoclonal antibody and the anti-6His tag monoclonal antibody, and there was no obvious difference in the Western blotting results.

[0080] 11. Indirect immunofluorescence identification of monoclonal antibody reactivity

[0081] Chicken embryo fibroblasts (CEF) were inoculated in a 96-well cell culture plate at a cell density of 80%, and 5 pfu of MDV / RB1B strain was added to each well after 24 hours. After 96 hours of culture, the cells were fixed on ice for 15 minutes with 4% tissue cell fixing solution, and then permeabilized on ice for 15 minutes with 0.1% triton X-100. After 1 hour of blocking with 5% BSA, the cells were washed with PBST for 4 times. The anti-MDV US3 protein monoclonal antibody and the polyclonal antibody (i.e. the serum of the 23# mouse described above) were diluted at a ratio of 1:1000 with 5% BSA, and they were used as the primary antibody, respectively, and incubated at room temperature for 2 hours. After washing with PBST for 4 times, goat anti-mouse IgG-FITC (Thermo Fisher, A11001) was diluted at a ratio of 1:100 with 5% BSA, and incubated at room temperature for 1 hour. After washing with PBST for 4 times, DAPI staining solution was diluted at a ratio of 1:1000 with PBS, and incubated at room temperature for 2 minutes. After washing with PBST for 4 times, the cells were observed and photographed under an inverted fluorescence microscope. As shown in Figure 8 , both the anti-MDV US3 protein monoclonal antibody ( Figure 8 C) and the polyclonal antibody ( Figure 8 B) could detect the US3 protein expressed in the CEF cells infected with the MDV / RB1B strain, and showed green fluorescence.

[0082] Chicken embryo fibroblasts (CEF) were inoculated in 24-well cell culture plates at a cell density of 80%. pcDNA3.1-HA-US3 plasmid carrying the HA-US3 gene (SEQ ID NO: 3) was transfected into CEF cells using Lipofectamine 3000 transfection reagent (Thermo Fisher, L3000015) according to the reagent use manual, and cultured for 48 h. Indirect immunofluorescence was performed using anti-MDV US3 protein monoclonal antibody and mouse anti-hemagglutinin (HA) tag antibody (Sigma, H9658) as primary antibodies, and goat anti-mouse IgG-FITC (Thermo Fisher, A11001) as secondary antibody, according to the above procedure. The results are shown in Figure 9 Fig. 2A, Fig. 2B and Fig. 2C, respectively. Anti-MDV US3 protein monoclonal antibody Figure 9 Fig. 2A, Fig. 2B and Fig. 2C, respectively. Anti-MDV US3 protein monoclonal antibody Figure 9 Fig. 2A, Fig. 2B and Fig. 2C, respectively. Anti-MDV US3 protein monoclonal antibody

[0083] 12. Specific detection of monoclonal antibody

[0084] The prepared 6His-MDV US3 protein, chicken gamma-interferon (Ch-IFNγ) shown in SEQ ID NO: 4, chicken interleukin 2 (Ch-IL-2) shown in SEQ ID NO: 5, Marek's disease virus phosphorylated protein 38 (MDV pp38) shown in SEQ ID NO: 6, Marek's disease virus gB protein (MDV gB) shown in SEQ ID NO: 7, and Marek's disease virus Meq protein (MDV Meq) shown in SEQ ID NO: 8 were diluted to 2.5 μg / mL, respectively. The above various proteins at a concentration of 2.5 μg / mL were added to a 96-well enzyme-labeled plate as antigens, 100 μL per well, and incubated at 4°C overnight. After washing with PBST for 5 times, 5% skim milk was added and incubated at 37°C for 2 h. After removing the blocking solution, anti-MDV US3 protein monoclonal antibody diluted with 5% skim milk at a concentration of 25 μg / mL was added, 100 μL per well, and incubated at 37°C for 1 h. After washing with PBST for 5 times, goat anti-mouse IgG-HRP (Sigma, 12-349) diluted with 5% skim milk at a dilution of 1:1000 was added, 100 μL per well, and incubated at 37°C for 45 min. After washing with PBST for 5 times, 100 μL of TMB single-component color developing solution was added to each well, and reacted at 37°C for 15 min in the dark. After adding 50 μL of ELISA stop solution to each well to terminate the reaction, the OD450nm value was determined.

[0085] The results showed that the anti-MDV US3 protein monoclonal antibody could specifically bind to 6His-MDV US3 protein and showed no cross-reactivity with Ch-IFNγ, Ch-IL-2, MDV pp38, MDV gB, and MDV Meq protein antigens. Figure 10 It has good specificity.

[0086] 13. Sensitivity testing of monoclonal antibodies

[0087] The anti-MDV US3 serum from mice #23 was purified using the Thermo Protein A / G purification system to obtain a polyclonal antibody against MDV US3 protein. Indirect ELISA was performed: 1 μg / mL of 6His-MDV US3 protein was added to each well of a 96-well ELISA plate as the antigen, 100 μL per well, and incubated overnight at 4°C. After washing five times with PBST, 5% skim milk was added, and the plate was blocked at 37°C for 2 h. The anti-MDV US3 monoclonal antibody and anti-MDV US3 polyclonal antibody were diluted to 1, 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.015625, and 0.0078125 μg / mL, respectively. Each dilution of the two antibodies was used as the primary antibody, and 100 μL was added to each well of the ELISA plate, incubated at 37°C for 1 h. After washing five times with PBST, add 100 μL of goat anti-mouse IgG-HRP (Sigma, 12-349) diluted 1:1000 with 5% skim milk to each well and incubate at 37°C for 45 min. Add 100 μL of TMB single-component chromogenic solution to each well and incubate at 37°C in the dark for 15 min. Stop the reaction by adding 50 μL of ELISA stop solution to each well and measure the OD450 nm value. Simultaneously, perform the above indirect ELISA detection using serum from unimmunized mice as the primary antibody as a negative control. Calculate the P / N value (sample OD450 nm value / negative control OD450 nm value), and consider a P / N ≥ 2.1 as positive.

[0088] The results are as follows Figure 11 As shown, the lowest concentration for a positive result in the detection of anti-MDV US3 protein monoclonal antibody is 0.03125 μg / mL, and the lowest concentration for a positive result in the detection of anti-MDV US3 protein polyclonal antibody is 1 μg / mL.

[0089] Example 2. Detection of Marek's disease virus (MDV) by direct immunofluorescence assay

[0090] 1. FITC labeling of monoclonal antibodies

[0091] Take out 5-isothiocyanate fluorescein (FITC) reagent 1 h in advance and restore to room temperature. Take 1.4 mg of anti-MDV US3 protein monoclonal antibody obtained in Example 1, add 0.05 M CBS•NaCl (0.8%) to the ultrafiltration tube, mix well, and centrifuge to make up to 140 μL. Weigh 0.21 mg of FITC, dissolve it, and add it to the anti-MDV US3 protein monoclonal antibody, mix well, and incubate at 4°C overnight in the dark. Transfer the above liquid from the centrifuge tube to a 14Kd dialysis bag, dialyze with PBS solution at 4°C in the dark for 24 h, and change the solution. Suck the liquid from the dialysis bag into a centrifuge tube, wrap the centrifuge tube with tin paper, add PBS to make up to the volume, add an equal volume of glycerol, and store at -20°C to obtain the FITC-labeled anti-MDV US3 protein monoclonal antibody.

[0092] 2. Direct immunofluorescence detection of MDV

[0093] Inoculate chicken embryo fibroblasts (CEF) into a 24-well cell culture plate at a cell density of 80%, and after 24 h, add 20 pfu of MDV / RB1B strain to each well. After 96 h of culture, fix the cells on ice with 4% tissue cell fixative for 15 min, and then add 0.1% triton X-100 for ice permeation for 15 min. Divide the permeated cells into two groups, one for direct immunofluorescence detection and the other for indirect immunofluorescence detection.

[0094] Direct immunofluorescence detection: Take the permeated cells, block with 5% BSA for 1 h, wash with PBST for 3 times, dilute the FITC-labeled anti-MDV US3 protein monoclonal antibody with 5% BSA at a ratio of 1:100, add to the sample well, 200 μL / well, and incubate at room temperature for 30 min. Wash with PBST for 3 times. Dilute the DAPI staining solution with PBS at a ratio of 1:1000, add to the sample well, 200 μL / well, and incubate at room temperature for 2 min. After washing with PBST for 3 times, observe the cells under a fluorescence microscope and take pictures.

[0095] Indirect immunofluorescence detection: Take the permeated cells, block with 5% BSA for 1 h, wash with PBST for 4 times. Dilute the anti-MDV US3 protein monoclonal antibody with 5% BSA at a ratio of 1:100, add to the sample well, 200 μL / well, and incubate at room temperature for 2 h. Wash with PBST for 4 times. Dilute goat anti-mouse IgG-FITC (Thermo Fisher, A11001) with 5% BSA at a ratio of 1:100, add to the sample well, 200 μL / well, and incubate at room temperature for 1 h. Wash with PBST for 4 times. Dilute the DAPI staining solution with PBS at a ratio of 1:1000, add to the sample well, 200 μL / well, and incubate at room temperature for 2 min. After washing with PBST for 4 times, observe the cells under a fluorescence microscope and take pictures.

[0096] The results show that there is no significant difference between the detection effect of the direct immunofluorescence method and the detection effect of the indirect immunofluorescence method using the anti-MDV US3 protein monoclonal antibody to detect the MDV US3 protein Figure 12 .

Claims

1. A monoclonal antibody against Marek's disease virus US3 protein, which is secreted by the hybridoma cell line with the preservation number of CGMCC No. 46324.

2. A hybridoma cell line secreting a monoclonal antibody against Marek's disease virus US3 protein, which has the preservation number of CGMCC No. 46324.

3. A kit for detecting Marek's disease virus US3 protein, which comprises the monoclonal antibody against Marek's disease virus US3 protein according to claim 1.

4. The kit of claim 3, wherein The kit further comprises general reagents for direct immunofluorescence method.

5. The kit of claim 4, wherein The general reagents comprise fluorescent substances for binding to the monoclonal antibody against Marek's disease virus US3 protein.

6. The kit according to claim 4 or 5, characterized in that, The fluorescent substances are fluorescein isothiocyanate.

7. The kit of claim 3, wherein The kit further comprises general reagents for indirect enzyme-linked immunosorbent assay or indirect immunofluorescence method.

8. The kit of claim 7, wherein The general reagents comprise signal marker labeled anti-mouse IgG antibody for binding to the monoclonal antibody against Marek's disease virus US3 protein.

9. A method of making a monoclonal antibody against a Marek's disease virus US3 protein comprising: The hybridoma cell line with the preservation number of CGMCC No. 46324 is injected into the abdominal cavity of mice, ascites is collected and the antibody is purified from the ascites.

10. Use of the monoclonal antibody against Marek's disease virus US3 protein according to claim 1 in the preparation of a product for detecting Marek's disease virus US3 protein.

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