A hybridoma cell line stably secreting anti-mink Aleutian disease virus monoclonal antibodies and its application

Abstract

The present invention discloses a hybridoma cell line that stably secretes monoclonal antibodies against mink Aleutian disease virus and its application, belonging to the field of biotechnology. The monoclonal antibody of the present invention is secreted by the hybridoma cell line with the deposit number CGMCC No. 23875. The hybridoma cell line has the ability to stably secrete IgG1 antibodies, and the monoclonal antibodies produced are high in titer and single in subclass, and can specifically recognize ADV VP2 protein. 492 FPHEV 496 The antigen epitope is 100% homologous to 24 ADV strains published domestically and internationally. It reacts specifically with ADV strains or proteins and can be used as a diagnostic reagent for mink Aleutian disease. It has practical application value and provides a material basis and technical support for clinical differential diagnosis and laboratory research of mink Aleutian disease.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a hybridoma cell line stably secreting anti-mink Aleutian disease virus monoclonal antibodies and applications thereof. Background Art

[0002] Aleutian mink disease (AD) is a chronic wasting infectious disease caused by the Aleutian mink disease virus (ADV). It is also known as plasmacytosis. It mainly attacks mink immune cells and is characterized by diffuse increase in plasma cells, decreased reproductive capacity, persistent viral infection and immune system disorders. It mainly leads to a serious decline in the reproductive capacity, immune level, fur development, etc. of infected minks. It is one of the three major viral diseases that endanger the mink farming industry.

[0003] ADV belongs to the Parvoviridae family and the Aleutian Parvovirus genus. It is a single-stranded linear negative-sense DNA virus that can replicate itself. The virus particles have no envelope, no sugars, and no lipids. The diameter is 22 to 25 nm and they are icosahedral symmetrical. Each virus particle contains 60 capsid particles, and the outer diameter of each capsid is 128A.

[0004] The structural protein VP2 is the primary immunogenic antigenic protein of ADV. It can neutralize the virus in vitro and serves as a carrier of antigenic determinants. It is closely related to the pathogenicity and host selection of the virus. The VP2 gene is the preferred gene for studying the structure and function of ADV virion proteins, the complex pathogenic mechanisms of ADV, and the development of ADV detection technologies. The VP2 protein is an ideal target antigen for detecting ADV-specific antibodies.

[0005] Aleutian disease in minks has a long incubation period, with a higher incidence rate in male minks than in females, and a higher infection rate in adult minks than in pups. The incidence and mortality rates in autumn and winter are higher than in spring and summer. The main sources of infection are sick minks and infected minks in the incubation period. The main routes of transmission of the virus within mink populations are horizontal and vertical transmission. Horizontal transmission primarily occurs through contamination of the mink farm environment by urine, excrement, and saliva from infected minks, which in turn infect healthy minks through contact. Vertical transmission primarily occurs through vertical infection of the placenta of infected female minks and postpartum breastfeeding, which infects pups. The virus can replicate in the female mink's placenta, and antibodies in the female mink's body are unable to block vertical transmission.

[0006] Mink Aleutian disease is a typical immune complex disease that mainly erodes the reticuloendothelial system, triggering the body's humoral immunity, causing diffuse proliferation of plasma cells and hypergammaglobulinemia. However, the antibodies produced not only fail to neutralize the virus, but instead combine with the virus to form immune complexes, helping the virus invade cells and triggering Fc receptor-mediated antibody-dependent enhancement. This is one of the reasons why the immune system function of minks infected with the Mink Aleutian disease virus is disrupted and their vaccination treatment fails.

[0007] Because mink Aleutian disease infects pups and adults differently, it presents different clinical symptoms. When pups are infected with mink Aleutian disease, the disease progresses rapidly, manifesting as symptoms of acute interstitial pneumonia such as respiratory distress, cough, and fever, with a high mortality rate. When adult minks are infected with mink Aleutian disease, they mainly show symptoms of a slowly progressive disease, specifically lethargy, loss of appetite, progressive weight loss, and a significantly increased appetite. In winter, they may chew on ice cubes, have unkempt and dull fur, experience severe anemia, black, coal-tar-like loose stools, oral ulcers, bleeding at the root of the tongue, and irritability. In the later stages of the disease, they develop obvious neurological symptoms, such as ataxia and hind limb paralysis. Death is often caused by secondary infections from other diseases and uremia. Female minks may miscarry or give birth to mummified fetuses, while male minks may have testicular hypoplasia and impaired reproductive capacity.

[0008] Due to the unique pathogenic mechanism of the Aleutian mink disease virus, there is currently no effective vaccine or specific treatment for the disease. Mink-farming regions around the world use seed purification and positive culling methods to control the disease. The homology of the ADV strains circulating in various provinces in my country varies, and a universal detection technology for different ADV strains is currently lacking.

[0009] The preparation of monoclonal antibodies has laid the foundation for immunological diagnosis of Aleutian disease in mink, further enriching the development of monoclonal antibody-based detection methods such as ELISA and colloidal gold test strips. Using monoclonal antibodies, ADV detection technology will develop towards greater speed, simplicity, sensitivity, specificity, high throughput, and automation.

[0010] Antigenic epitopes are the material basis of immune responses and determine the specificity of monoclonal antibodies and the scope of application of detection methods. However, there are few studies on the antigenic epitopes of ADV monoclonal antibodies. In 2016, Yi et al. prepared a specific monoclonal antibody 1M13 against the peptide segment 291-502 of ADV VP2 protein. By performing ELISA analysis on a series of partially overlapping synthetic peptides, they determined that 386 HLQQNFSTRYIYD 398It is the smallest linear B cell epitope that can be recognized by monoclonal antibody 1M13 (Yi et al., 2016); in 2018, Lu et al. prepared a specific monoclonal antibody 1G5 against the 200-588 peptide segment of ADV VP2 protein and confirmed that the smallest linear B cell epitope it recognized is located at 459 EEEGWPAASGTHFED 473 However, these two epitopes are not conserved in most strains of ADV both domestically and internationally, and the homology among ADV strains isolated in my country in recent years cannot reach 100%, which still cannot effectively solve the problem of ADV detection in my country. Summary of the Invention

[0011] In response to the above-mentioned prior art, the present invention aims to provide a hybridoma cell line that stably secretes monoclonal antibodies against mink Aleutian disease virus and its use. The monoclonal antibodies secreted by the hybridoma cell line of the present invention recognize highly conserved antigenic epitopes, sharing 100% homology with representative ADV strains both domestically and internationally. The amino acid sequence has not undergone genetic mutations during the course of the virus's prevalence, enabling the identification of a wider range of ADV strains both domestically and internationally, and possessing potential application in the development of new ADV diagnostic tools.

[0012] To achieve the above object, the present invention adopts the following technical solutions:

[0013] In a first aspect, the present invention provides a hybridoma cell line, the deposit number of which is CGMCC No. 23875.

[0014] The hybridoma cell line of the present invention can stably secrete the monoclonal antibody ADV-GZ2104-H7 against ADV VP2 protein, and its nuclear chromosome number is 102. The hybridoma cell line was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on November 19, 2021, and was classified and named as anti-ADV monoclonal cell line, with the deposit number CGMCC No. 23875. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0015] The use of the above hybridoma cell line in the preparation of monoclonal antibodies that recognize the VP2 protein of mink Aleutian disease virus also falls within the scope of protection of the present invention.

[0016] The second aspect of the present invention provides a monoclonal antibody secreted and produced by the hybridoma cell line with the above-mentioned deposit number CGMCC No. 23875.

[0017] The monoclonal antibody secreted by the hybridoma cell line with the deposit number CGMCC No. 23875 of the present invention has the following characteristics:

[0018] (1) The antibody subclass of the monoclonal antibody is IgG1.

[0019] (2) The monoclonal antibody can specifically recognize mink Aleutian disease virus, but does not recognize mink enteritis parvovirus and mink canine distemper virus, and has good specificity and reactivity.

[0020] (3) The monoclonal antibody can specifically react with ADVVP2 prokaryotic expression protein and natural viral protein.

[0021] (4) The ascites antibody titer of the monoclonal antibody reached 1:1.6×10 6 , high potency.

[0022] (5) The shortest linear epitope specifically recognized by the monoclonal antibody is ADV VP2 protein 492 FPHEV 496 The location of this epitope is based on the VP2 gene of the standard strain of mink Aleutian disease virus (ADV-G), Genbank: M20036. This epitope is located on the protein's surface, facilitating direct contact with antibodies, thereby facilitating an immune response. Furthermore, this epitope is highly conserved among ADV strains, sharing 100% homology with 24 representative ADV strains reported domestically and internationally. This makes it a promising target for detecting the spread of ADV strains, helping to improve the specificity and universality of ADV detection technology and accelerate its purification.

[0023] The third aspect of the present invention provides the use of the hybridoma cell line or monoclonal antibody in the preparation of a product for detecting mink Aleutian disease virus.

[0024] In the above applications, preferably, the product can be an ELISA kit, a colloidal gold test strip, an indirect immunofluorescence assay (IFA) kit or other immunofluorescence detection kit.

[0025] The fourth aspect of the present invention provides the use of the above-mentioned monoclonal antibody in the preparation of a drug for preventing or treating mink Aleutian disease.

[0026] A fifth aspect of the present invention provides a kit for detecting mink Aleutian disease virus, which contains an effective amount of the above-mentioned monoclonal antibody.

[0027] Beneficial effects of the present invention:

[0028] (1) Based on the ADV-G strain gene sequence published in NCBI (GenBank: M20036), the present invention extracts a region of the VP2 gene that is more representative (higher homology), has a higher antigenic index, and expresses 184 amino acids as an immunogen. A hybridoma cell line is obtained through cell fusion, multiple screenings, and cloning. The hybridoma cell line of the present invention has a chromosome number of 102, which is consistent with the chromosome number of hybridoma cells. The hybridoma cell line of the present invention can stably secrete antibodies when continuously passaged for 10 generations under the same conditions. After freezing and thawing, the antibody secretion capacity does not decrease for at least 12 months, indicating good stability.

[0029] (2) The monoclonal antibodies secreted by the hybridoma cell line of the present invention can specifically recognize mink Aleutian disease virus and have good specificity and reactivity; the ascites antibody titer is high; the shortest linear epitope that can be recognized is ADV VP2 protein 492 FPHEV 496 This antigen epitope has 100% homology with 24 ADV strains published at home and abroad, making it a good target for detecting the spread of ADV strains. It helps to improve the specificity and universality of ADV detection technology and accelerate the purification of ADV. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Recombinant expression plasmid map; among them, Figure A is the recombinant plasmid pET-32a-ADV map, and Figure B is the recombinant plasmid pGEX-6P-1-ADV map.

[0031] Figure 2 : Single and double enzyme digestion identification results of recombinant expression plasmids; in Figure A, lane 1 is the double enzyme digestion identification result of pET-32a-ADV recombinant plasmid using BamHI and NotI; lane 2 is the BamHI single enzyme digestion identification result of pET-32a-ADV recombinant plasmid; in Figure B, lane 1 is the double enzyme digestion identification result of pGEX-6P-1-ADV recombinant plasmid using BamHI and NotI; lane 2 is the BamHI single enzyme digestion identification result of pGEX-6P-1-ADV recombinant plasmid.

[0032] Figure 3 : Prokaryotic expression, purification and identification of His-ADV VP2 recombinant protein; Figure A: 1, protein expressed by the empty vector in E. coli; 2, expression product of the recombinant plasmid in E. coli without IPTG induction; 3, expression product of the recombinant plasmid in E. coli after IPTG induction; 4, purified His-ADV VP2 recombinant protein; Figure B: 1, Western blot identification results using an anti-His tag monoclonal antibody as the primary antibody.

[0033] Figure 4: Prokaryotic expression, purification and identification of GST-ADV VP2 recombinant protein; Figure A: 1, protein expressed by empty vector in E. coli; 2, expression product of recombinant plasmid in E. coli without IPTG induction; 3, expression product of recombinant plasmid in E. coli after IPTG induction; 4, purified GST-ADV VP2 recombinant protein; Figure B: 1, Western blot identification results with the primary antibody being anti-GST tag monoclonal antibody.

[0034] Figure 5 : Chromosome number of hybridoma cell line ADV-GZ2104-H7.

[0035] Figure 6 : Preparation of ascites antibodies.

[0036] Figure 7 : Subclass identification results of monoclonal antibody ADV-GZ2104-H7.

[0037] Figure 8 : IFA identifies the specificity of monoclonal antibodies; among them, Figure A is the ADV-SD strain, Figure B is the ADV-SD1908 strain, Figure C is the ADV strain isolated from a mink farm in Zhucheng, Shandong, Figure D is the ADV strain isolated from a mink farm in Haiyang, Shandong, Figure E is the ADV strain isolated from a mink farm in Rizhao, Shandong, Figure F is the positive control (positive serum of BALB / c mice immunized with His-ADV VP2 recombinant protein as the primary antibody to recognize CRFK cells infected with ADV-SD strain), Figure G is the MEV strain, Figure H is the CDV strain, and Figure I is the negative control.

[0038] Figure 9 : Western blot identification of monoclonal antibodies, among which, Figure A shows the reactivity identification of the monoclonal antibody with the prokaryotic expressed protein His-ADVVP2 antigen (lane 1 is the His-ADV VP2 recombinant protein, and lane 2 is the His-tagged protein); Figure B shows the reactivity identification of the monoclonal antibody with the prokaryotic expressed protein GST-ADV VP2 antigen (lane 1 is the GST-ADV VP2 recombinant protein, and lane 2 is the GST-tagged protein); Figure C shows the reactivity identification of the monoclonal antibody with the whole viral protein antigen in CRFK cells (lane 1 is the whole protein of the infected cells, and lane 2 is the whole protein of the uninfected cells); Figure D shows the reactivity identification of the monoclonal antibody with the whole viral protein antigen of the mink disease material (lane 1 is the positive mink disease material in Rizhao, Shandong, lane 2 is the positive mink disease material in Wendeng, Shandong, lane 3 is the positive mink disease material in Zhucheng, Shandong, lane 4 is the positive mink disease material in Haiyang, Shandong, and lane 5 is the negative mink disease material).

[0039] Figure 10 : Identification of ascites antibody titers.

[0040] Figure 11 : Identification of antibody secretion stability; Figure A shows the antibody secretion stability of hybridoma cells that have been continuously passaged for 10 generations; Figure B shows the antibody secretion stability of hybridoma cells that have been regularly revived.

[0041] Figure 12 : Identification of antigen epitopes recognized by monoclonal antibodies; Figure A shows the segmented expression design of ADV VP2 protein; Figure B shows the results of immunoblotting analysis of expressed antigens and monoclonal antibodies.

[0042] Figure 13 : ELISA accurately identifies antigen epitopes.

[0043] Figure 14 : Prediction of antigen epitope secondary structure; Figure A is the secondary structure analyzed by DNAstar software; Figure B is the secondary structure analyzed by SOPMA software.

[0044] Figure 15 : Three-dimensional conformation analysis of the antigen epitope; Figure A is the rod-shaped three-dimensional structure of the antigen epitope; Figure B is the globular three-dimensional structure of the antigen epitope.

[0045] Figure 16 : Analysis of antigenic epitope conservation. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0047] As previously mentioned, mink Aleutian disease is a typical immune complex disease. After infecting the host, the virus causes persistent infection and slow replication, leading to significant antibody-dependent enhancement (ADAE). This makes the development of effective vaccines and medications difficult, and the disease can only be controlled through quarantine and elimination strategies. Due to the varying homology of ADV strains circulating in various provinces in my country, there is currently a lack of universal detection technology for different ADV strains.

[0048] The preparation of monoclonal antibodies has laid the foundation for the immunological diagnosis of Aleutian disease in mink. The antigenic epitope is the material basis of the immune response and determines the specificity of the monoclonal antibody and the scope of application of the detection method. However, there is currently little research on the antigenic epitopes of ADV monoclonal antibodies. Furthermore, the antigenic epitopes recognized by the currently reported ADV monoclonal antibodies are not conserved among most ADV strains both domestically and internationally. Furthermore, the homology among ADV strains isolated in my country in recent years has not yet reached 100%, which still poses a significant challenge to ADV detection in my country.

[0049] In view of this, the present invention has conducted in-depth research on monoclonal antibodies that can universally recognize different ADV strains. The antigenic epitope recognized by the monoclonal antibody is first determined by the amino acid sequence of the immunogen used in the preparation of the hybridoma. However, not all amino acids can form antigenic epitopes. Only those structural regions with a high antigenic index recognized by antigen-presenting cells and a large difference in the histocompatibility antigen complex MHC1 can become antigenic epitopes. In the immunogen used, structural fragments that meet these characteristics have a certain percentage of becoming antigenic epitopes. Due to the randomness during fusion and screening, it is impossible to say with certainty whether they are determined in advance or are random. In actual research, it is necessary to optimize and shorten the antigenic epitope to be screened as an immunogen. Reducing subsequent randomness is a scientific strategy for preparing monoclonal cells that specifically recognize antigenic epitopes. In this study, based on the ADV-G strain gene sequence published in NCBI (GenBank: M20036), a region of the VP2 gene with a broad representation (high homology) and a high antigenic index was extracted, expressing 184 amino acids as an immunogen. This immunogen was co-administered with Freund's adjuvant and inoculated into BALB / c mice four times, 15 days apart. Splenocytes from these immunized mice were fused with mouse myeloma cells (SP2 / 0 cells). Hybridomas were screened using HAT / HT medium and then screened for antibodies against ADV using ELISA and indirect immunofluorescence assays (IFA). Multiple cloning and purification of the hybridomas using the limiting dilution method ultimately resulted in the isolation of a single hybridoma cell line that stably secretes a monoclonal antibody against the ADV VP2 protein.

[0050] The monoclonal antibody secreted by the hybridoma cell line of the present invention recognizes an antigenic epitope that is highly conserved among ADV strains isolated and published at home and abroad, and has 100% homology with 24 representative ADV strains selected, indicating that the monoclonal antibody of the present invention has the ability to recognize and bind to all these strains. The detection method established using this antibody may be able to detect ADV strains that include all currently known strains, and its application value and advantages are obvious.

[0051] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below with reference to specific examples. If the specific experimental conditions are not specified in the examples, they are usually based on conventional conditions or the conditions recommended by the reagent company; the reagents and consumables used in the following examples, unless otherwise specified, can be obtained through commercial channels.

[0052] Example 1: Preparation of hybridoma cell lines

[0053] 1. Preparation of protein for immunization:

[0054] According to the ADV-G strain gene sequence published in NCBI (GenBank: M20036), the highly immunoreactive region 376 to 559aa of the VP2 gene was cut as the immunogen, and primers were designed for PCR amplification to obtain the amplified product.

[0055] The amplified product, pET-32a empty vector plasmid, and pGEX-6P-1 empty vector plasmid were double-digested with BamHI and NotI, and the digested products were recovered by gel gel and ligated into E. coli BL21 (DE3) competent cells. They were spread on LB solid medium containing ampicillin (50 μg / mL) and cultured at 37°C for 8-10 hours. Single colonies were randomly picked, shaken and cultured, and plasmids were extracted. Plasmids identified as positive by double enzyme digestion were sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequence determination. The results are as follows: Figure 1 、 2 As shown, the recombinant plasmids identified as correct by sequence determination were named pET-32a-ADV and pGEX-6P-1-ADV.

[0056] The bacterial solution containing the pET-32a-ADV recombinant plasmid was shaken and cultured until the OD 600 The pH value is about 0.6 to 0.8, so that E. coli is in the logarithmic growth phase, IPTG is added to a final concentration of 1mM, and the culture is shaken for 4 hours. The bacterial precipitate is collected and resuspended with PBS, and ultrasonically broken until the resuspended bacterial solution is transparent and not viscous. The supernatant is discarded and the inclusion body protein is collected. After purification by Ni-NTA affinity chromatography, SDS-PAGE electrophoresis is performed. The results are as follows Figure 3 To identify the prepared His-ADV VP2 protein, Western blot analysis was performed using an anti-His tag protein antibody. The results are shown in Figure 1. Figure 3 As shown in Figure B, there is a clear immune reaction band at the position of 50 kDa, indicating that the His-ADV VP2 protein for immunizing mice was successfully prepared.

[0057] The bacterial solution containing the pGEX-6P-1-ADV recombinant plasmid was expanded and induced at low temperature at 16°C for 16 hours. The bacterial solution was centrifuged and the precipitate was collected. The GST tag can make the target protein soluble in E. coli and easily maintain the complete activity of the target protein. Therefore, the precipitate was discarded, and the supernatant was obtained by centrifugation after sonication. It was purified by GST-tag Purification Resin affinity chromatography medium and then subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 4 As shown in A, in order to identify the prepared GST-ADV VP2 protein, it was identified and analyzed by Western blot mediated by anti-GST tag protein antibody. The results are shown in FIG. Figure 4As shown in Figure B, there is a clear immunoreactive band at the position of 48 kDa, indicating that the GST-ADV VP2 protein for screening positive hybridoma cell lines was successfully prepared.

[0058] 2. Animal immunization:

[0059] The purified His-ADV VP2 recombinant protein was injected subcutaneously at multiple points on the back of the neck to immunize 6-week-old female BALB / c mice. The immunizations were repeated four times, with two weeks between each immunization. For the first immunization, the recombinant protein was mixed with equal volumes of complete Freund's adjuvant and emulsified. For the second and third immunizations, the recombinant protein was emulsified with equal volumes of incomplete Freund's adjuvant. Seven days after the third immunization, blood was collected from the submandibular vein of the mice, and serum was separated. The mouse serum antibody titer was detected using an ELISA plate coated with GST-ADV VP2 recombinant protein. The antibody titer reached 1:10. 5 Mice were immunized with ADV VP2 protein (without adjuvant) (100 μg / mouse) for four booster doses. Three days later, spleen cells of the immunized mice were fused with SP2 / 0 (myeloma cells).

[0060] 3. Preparation of feeder cells:

[0061] One day before cell fusion, mouse peritoneal macrophages were taken to prepare feeder layer cells. Non-immunized BALB / c mice were taken, killed, and then immersed in 75% alcohol for disinfection. The mice were transferred to a clean bench, their limbs were spread out and fixed, and the abdominal skin was cut open with sterile scissors and tweezers to fully expose the peritoneum. Culture medium was injected into the peritoneal cavity with a sterile syringe, and the abdomen was repeatedly pressed for 1 minute to fully free the macrophages. The culture medium containing peritoneal macrophages was then aspirated; after counting the cells, the cell concentration was maintained at 1×10 5 The cell suspension was added to a 96-well plate and observed for contamination. If there was no contamination, it could be used for fusion experiments.

[0062] 4. Preparation of myeloma cells:

[0063] One week before cell fusion, resuscitate and culture SP2 / 0 cells to adjust their cell state. Perform cell fusion when the cells are in the logarithmic growth phase, have a high cell density, and are round and translucent. Gently remove the SP2 / 0 cells from the flask wall using DMEM basal medium and transfer them to a sterile centrifuge tube. Centrifuge at 1,000 rpm for 8 minutes, discard the supernatant, and wash the cells again with DMEM basal medium. Finally, resuspend the cells in 10 mL of DMEM basal medium. Count the cells using trypan blue staining and set aside.

[0064] 5. Preparation of splenocytes:

[0065] BALB / c mice that had been boosted with immunization were enucleated, blood was collected, and serum was separated. The mice were killed by cervical dislocation and soaked in 75% alcohol for 10 minutes. The abdominal cavity of the mice was opened in a clean bench, and the spleen of the mice was aseptically removed. The membrane connective tissue was removed, and the spleen was ground on a 70 μm cell filter to prepare a single-cell suspension. The suspension was centrifuged at 1,000 rpm for 10 minutes, and the supernatant was discarded. The pellet was resuspended in 10 mL of DMEM basal medium and mixed. The cells were counted with trypan blue staining solution and set aside.

[0066] 6. Cell fusion:

[0067] At a ratio of 5:1 splenocytes:SP2 / 0 cells, thoroughly mix an appropriate amount of SP2 / 0 cells and splenocytes in a sterile 50mL centrifuge tube. Centrifuge and discard the supernatant. Gently tap the bottom of the tube to loosen and evenly distribute the cell pellet. Slowly and evenly add 1mL of 50% PEG over 1 minute at 37°C and let it stand for 1 minute. Then, slowly and evenly add DMEM basal medium to terminate fusion. Centrifuge and discard the supernatant. Slowly add HAT complete medium, gently blow off the pellet and mix thoroughly. The mixed cell suspension is then added dropwise to five 96-well culture plates containing feeder cells prepared above. Incubate in a 37°C, 5% CO2 incubator. Observe the cell growth status in the culture plates and save the cell supernatant for hybridoma screening.

[0068] 7. Screening, identification and cloning culture of positive hybridoma cells:

[0069] Positive hybridoma cell lines were screened using the indirect ELISA detection method. The ELISA plate was coated with three proteins: His-ADV VP2 protein, GST-ADVVP2 protein, and purified pET-32a empty vector protein (His-tagged protein). The cell supernatant was used as the primary antibody, the positive serum of BALB / c mice was used as the positive control, and the supernatant of SP2 / 0 cells was used as the negative control. The wells that reacted positively with His-ADV VP2 and GST-ADV VP2 but negatively with the His-tagged protein were selected as the positive clones.

[0070] Positive hybridoma wells identified by indirect ELISA were screened again using IFA to ensure antibody specificity. Hybridomas that tested positive by both ELISA and IFA were subcloned at least three times using limiting dilution. The following steps were performed: hybridoma cells in positive wells were counted, diluted with HT medium, and plated into a 96-well plate containing feeder cells, ensuring only a single hybridoma cell per well. After multiple subcloning and screening steps, a hybridoma cell line that stably secreted anti-ADV monoclonal antibodies was obtained and designated ADV-GZ2104-H7.

[0071] The colchicine method was used to detect the chromosome number of hybridoma cells. Chromosome analysis of hybridoma cells is one of the objective criteria for obtaining true hybridoma cells. The chromosome number of each complete hybridoma cell should be the sum of the chromosome numbers of mouse spleen cells and SP2 / 0 cells. Colchicine can destroy the spindle fibers of the cells and obtain metaphase cells. The cells are then hypotonic treated with potassium chloride solution (0.075 mol / L) to increase the volume of the entire cell and make the chromosomes in the cells loose. After hypotonic treatment, the cells are fixed with methanol-glacial acetic acid solution, stained with 10% Giemsa stain, and observed under a microscope (1000x oil lens). Cells with non-overlapping, well-dispersed, and non-lost chromosomes are selected for counting. The results are as follows: Figure 5 As shown, the chromosome number of hybridoma cell line ADV-GZ2104-H7 is 102.

[0072] The hybridoma cell ADV-GZ2104-H7 was deposited in the General Microbiology Center of the China Culture Collection Administration on November 19, 2021. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 23875, and the classification name is anti-ADV monoclonal cell line.

[0073] Example 2: Preparation of ascites antibodies using hybridoma cells

[0074] (1) Female BALB / c mice (12 weeks old) in good condition were selected, and the hybridoma cell line prepared in Example 1 was pipetted into a single cell suspension and the suspension was diluted to 1×10 6 Hybridoma cell suspension was injected intraperitoneally at a dose of 100mg / histogram. The cells were inoculated for about 10 days. Figure 6 As shown, the mouse abdomen was obviously distended and the ascites was slowly extracted.

[0075] (2) The collected ascites was centrifuged at 12,000 rpm for 10 min at 4°C to remove the upper layer of fat and the bottom cellular components and other precipitates. The supernatant was collected and filtered through a 0.45 μm filter to obtain the purified ascites antibody, which is the monoclonal antibody ADV-GZ2104-H7.

[0076] Example 3: Biological Characterization of Monoclonal Antibodies

[0077] (1) Monoclonal antibody subclass identification

[0078] The monoclonal antibody obtained in Example 2 was subjected to subclass identification according to the instructions of the Sino Biological Isotyping Kit for Mouse Monoclonal Antibody. Figure 7As shown, the antibody subclass of the monoclonal antibody ADV-GZ2104-H7 prepared in the present invention is IgG1.

[0079] (2) Identification of virus recognition characteristics of monoclonal antibodies

[0080] Indirect immunofluorescence (IFA) was used to detect the specificity of the ascites antibody prepared in Example 2, and the steps were as follows:

[0081] CRFK cells in the logarithmic growth phase were plated into 24-well cell culture plates. When the cells formed a monolayer, they were inoculated with five ADV strains and one mink enteritis virus (MEV) strain isolated and preserved in our laboratory. African green monkey kidney (Vero) cells in the logarithmic growth phase were plated into 24-well cell culture plates. When the cells formed a monolayer, they were inoculated with a mink distemper virus (CDV) strain. Uninoculated CRFK cells served as a negative control.

[0082] When the infected cells show cytopathic effect (CPE), discard the cell culture medium; wash with PBS three times, 5 minutes each time; fix with 4% paraformaldehyde at 4°C for 20 minutes, wash with PBS three times, 5 minutes each time; add 1:1000 diluted ascites antibody, incubate at 37°C for 1 hour, wash with PBS three times, 5 minutes each time; add 1:100 diluted FITC-labeled goat anti-mouse IgG fluorescent secondary antibody, incubate at 37°C for 1 hour, wash with PBS three times, 5 minutes each time; finally, observe the cell membrane and cytoplasm under an inverted fluorescence microscope in a light-proof environment, and those with green fluorescence are positive.

[0083] The results are as follows Figure 8 As shown, the ascites antibody prepared in Example 2 can specifically react with 5 isolated ADV strains, which is the same as the positive control result. Green fluorescence appears on the positive cell membrane and cytoplasm. It reacts negatively with MEV-infected CRFK cells, CDV-infected Vero cells and uninfected CRFK cells, with no fluorescence. It proves that the monoclonal antibody ADV-GZ2104-H7 prepared by the present invention can specifically recognize mink Aleutian disease virus, but does not recognize mink enteritis parvovirus and mink canine distemper virus, and has good specificity and excellent clinical applicability.

[0084] (3) Identification of viral protein recognition characteristics of monoclonal antibodies

[0085] Western blot was used to identify the reactivity of monoclonal antibodies to ADV VP2 protein expressed in vitro:

[0086] The prokaryotic expressed ADV VP2 recombinant protein was subjected to SDS-PAGE electrophoresis, and then the protein was transferred to a PVDF membrane; it was blocked with 5% skim milk powder at 4°C overnight, washed 4 times with TBST, each time for 8 minutes; a 1:1000 dilution of ascites antibody was added, incubated at room temperature for 2 hours, and washed 4 times with TBST, each time for 8 minutes; a 1:8000 dilution of HRP-labeled goat anti-mouse IgG secondary antibody was added, incubated at room temperature for 1 hour, and washed 4 times with TBST, each time for 8 minutes. The membrane was developed with an ultrasensitive ECL chemiluminescence kit and exposed to a protein developer to display protein bands. The results are as follows. Figure 9 As shown in A, the monoclonal antibody ADV-GZ2104-H7 prepared by the present invention can react specifically with the prokaryotic expressed His-ADV VP2 protein, and the band is consistent with the expected result, which is 50kDa; Figure 9 As shown in Figure B, the monoclonal antibody ADV-GZ2104-H7 can specifically react with the prokaryotic expressed GST-ADV VP2 protein, and the band is consistent with the expected result, which is 48 kDa.

[0087] Western blot analysis of the monoclonal antibody reacting with the whole virus VP2 protein antigen in CRFK cells:

[0088] Cat kidney cells (CRFK) were plated into 6-well cell culture plates, and ADV strains were inoculated when the cells grew into a monolayer. Uninfected CRFK cells were used as negative controls. When the infected cells showed cytopathic effect (CPE), samples were collected, and the CRFK cell pellets inoculated with ADV virus and the uninfected CRFK cell pellets were treated with RIPA lysis buffer and subjected to SDS-PAGE electrophoresis. The proteins were then transferred to PVDF membranes; blocked with 5% skim milk powder overnight at 4°C, washed with TBST 4 times, 8 minutes each time; ascites antibody diluted 1:1000 was added, incubated at room temperature with shaking for 2 hours, washed with TBST 4 times, 8 minutes each time; HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:8000 was added, incubated at room temperature with shaking for 1 hour, washed with TBST 4 times, 8 minutes each time, and developed with an ultrasensitive ECL chemiluminescence kit. Protein bands were displayed on a protein developer. The results are shown in Figure 2. Figure 9 As shown in Figure C, the monoclonal antibody ADV-GZ2104-H7 prepared by the present invention can specifically react with ADV viral protein with a band size of approximately 35 kDa, but does not react with negative control cells.

[0089] Identification of the reactivity of monoclonal antibodies with the whole virus protein antigen of positive ADV mink disease material:

[0090] Four mink Aleutian disease virus-positive mink disease samples and one mink Aleutian disease virus-negative mink disease sample collected and identified from Rizhao, Shandong, Wendeng, Shandong, Zhucheng, Shandong, and Haiyang, Shandong were collected and identified. The livers of the five disease samples were treated with RIPA lysis buffer, and the total protein of the mink disease samples was extracted for SDS-PAGE electrophoresis. The proteins were then transferred to PVDF membranes; blocked with 5% skim milk powder overnight at 4°C, washed with TBST 4 times, 8 minutes each time; added 1:1000 diluted ascites antibody, incubated at room temperature with shaking for 2 hours, washed with TBST 4 times, 8 minutes each time; added 1:8000 diluted HRP-labeled goat anti-mouse IgG secondary antibody, incubated at room temperature with shaking for 1 hour, washed with TBST 4 times, 8 minutes each time, developed with an ultrasensitive ECL chemiluminescence kit, and exposed to a protein developer to display protein bands. The results are as follows Figure 9 As shown in D, the monoclonal antibody ADV-GZ2104-H7 prepared by the present invention can specifically bind to the natural viral antigens extracted from the liver of ADV-positive minks in lanes 1, 2, 3, and 4, with a band size of approximately 35 kDa, and does not react with the protein extracted from the negative mink disease material, demonstrating that the prepared monoclonal antibody has good activity and is suitable for application in clinical detection of ADV strains.

[0091] (4) Monoclonal antibody titer

[0092] The titer of the ascites antibody prepared in Example 2 was detected by indirect ELISA method, and the steps were as follows:

[0093] The purified GST-ADV VP2 protein was used as the antigen to coat the 96-well ELISA plate, and the ascites antibody diluted in proportion was added as the primary antibody, the negative ascites prepared from SP2 / 0 cells was used as the control, and HRP-labeled goat anti-mouse IgG was used as the secondary antibody. In the dark, TMB single-component substrate color solution and stop solution were added to stop the color development, and the OD value was 0. 450nm Read the data and determine the ascites antibody titer. Figure 10 As shown in Figure 4, the titer of the ascites antibody prepared in Example 4 was 1:1638400 (approximately 1:1.6×10 6 ).

[0094] (5) Determination of antibody secretion stability

[0095] The hybridoma cell line ADV-GZ2104-H7 prepared in Example 1 was passaged for 10 generations under the same conditions, and the cell supernatant was detected by indirect ELISA coated with GST-ADV VP2 recombinant protein to identify the antibody secretion stability.

[0096] The results are as follows Figure 11 As shown in Figure A, the hybridoma cell line ADV-GZ2104-H7 was continuously passaged for 10 generations under the same conditions, and the hybridoma cells were able to stably secrete antibodies.

[0097] The hybridoma cell line ADV-GZ2104-H7 prepared in Example 1 was frozen and revived at 1, 3, 6, 9, and 12 months, respectively. After resuscitation, the cells were passaged at least 3 times. The cell growth status was observed under a microscope, and the antibody titer of the cell supernatant was determined by indirect ELISA.

[0098] The results are as follows Figure 11 As shown in Figure B, after the hybridoma cell line ADV-GZ2104-H7 was revived from cryopreservation, the antibody secretion ability did not decrease for at least 12 months, indicating good stability.

[0099] (6) Identification of monoclonal antibody-recognized antigen epitopes

[0100] In order to locate the antigen epitope recognized by the monoclonal antibody ADV-GZ2104-H7, a series of overlapping truncated fragments were designed, totaling 18 segments ( Figure 12 A) The protein was ligated to the pET-32a expression vector via BamHI and HindIII. The protein was expressed in prokaryotes and purified. Western blot and indirect ELISA were used to identify the epitope recognized by the monoclonal antibody.

[0101] The results are as follows Figure 12 As shown in B, all 18 truncated proteins can react with the primary antibody of His tag antibody, indicating that all 18 truncated proteins are correctly expressed. The monoclonal antibody reacts with the six segments of ADV 1-1 to ADV 1-6 in the first segmentation, and each segment overlaps with each other by 10 amino acids. The results show that the monoclonal antibody only reacts with ADV 1-4; then a second truncation is performed, and ADV 1-4 is truncated into six segments of ADV2-1 to ADV 2-6, and each segment overlaps with each other by 6 amino acids. The results show that the monoclonal antibody reacts with ADV 2-5 and ADV 2-6 at the same time, indicating that the antigen epitope recognized by the monoclonal antibody is located in the overlapping part of ADV2-5 and ADV2-6 proteins; then a third small-scale truncation is performed, and the overlapping part of ADV 2-5 and ADV 2-6 proteins is truncated to ADV 3-1 to ADV 3-6. The results show that the monoclonal antibody binds to ADV 3-1 ( 492 FPHEV 496 )、ADV 3-3( 491 EFPHEV 496 )、ADV 3-4( 491 EFPHEVL 497 )、ADV 3-5( 490 LEFPHEVL 497 )、ADV 3-6( 491 EFPHEVLD 498 ), does not bind ADV 3-2( 491EFPHE 495 ), indicating that the valine V at position 496 is a key amino acid constituting the antigenic epitope. If the valine V at position 496 is deleted, the structure of the antigenic epitope is destroyed, the protein loses its antigenicity, and cannot react with the antibody; while the presence or absence of glutamic acid E at position 491 does not affect the antigenicity of the protein, indicating that glutamic acid E at position 491 is not a key amino acid constituting the antigenic epitope. Therefore, through three truncations, it was preliminarily determined that the linear epitope recognized by the monoclonal antibody ADV-GZ2104-H7 is 492 FPHEV 496 .

[0102] In order to accurately locate the shortest antigen epitope recognized by the monoclonal antibody, the end-addition and subtraction peptide synthesis method was used to gradually reduce and increase amino acids from the N-terminus and C-terminus of the initially located amino acid sequence to synthesize five peptide segments. The antigen epitope was identified using the indirect ELISA method until the shortest antigen epitope was identified.

[0103] The results are as follows Figure 13 As shown in the results, when the phenylalanine F at position 492 is missing, the antigenicity of the epitope changes significantly. When the valine V at position 496 is missing, the antigenicity of the epitope also changes significantly, indicating that the phenylalanine F at position 492 and the valine V at position 496 are the key amino acids constituting this linear epitope. The ELISA results show that 492 FPHEV 496 On the basis of the above, a glutamic acid E is added to the N-terminus of the peptide or a leucine L is added to the C-terminus of the peptide, OD 450nm The absorbance values ​​did not change significantly. These results further confirm that: 492 FPHEV 496 It is the shortest linear epitope recognized by monoclonal antibody ADV-GZ2104-H7.

[0104] (7) Bioinformatics analysis of monoclonal antibody recognition of antigen epitopes

[0105] 1) Secondary structure analysis of antigenic epitopes

[0106] The distribution of antigenic epitopes is related to the secondary structure of proteins. DNAstar and SOPMA software were used to analyze the secondary structure of antigenic epitopes to improve the reliability of the results. SOPMA, the Gamier-Robson method, and the Chou-Fasman method predict secondary structure based on the likelihood of amino acid residues in a specific structure and the crystal structure of amino acids, respectively.

[0107] The results are as follows Figure 14 As shown, the antigen epitope 492 FPHEV 496The secondary structure of a protein is composed of α-helices and random coils. α-helices rise in a regular spiral around an axis, with a fixed shape and high stability. Random coils are protruding structures, mostly on the surface of proteins, with a loose structure, which is conducive to antibody chimera and easily forms antigenic epitopes.

[0108] 2) Three-dimensional conformation analysis of antigen epitopes

[0109] Antigen epitopes were constructed using the Swiss Model (https: / / swissmodel.expasy.org / ) online website. 492 FPHEV 496 The three-dimensional model was visualized using SPDBV software to analyze its spatial structure. The results are as follows Figure 15 As shown in the stick model, the antigen epitope 492 FPHEV 496 The five amino acids form a ring-shaped three-dimensional spatial conformation. The globular model shows that the five amino acids of the epitope are distributed on the surface of the protein, which is conducive to direct contact with antibodies, making it easier for the body to produce an immune response.

[0110] 3) Conservation analysis of antigenic epitopes

[0111] The 24 most representative ADV strains from the past 50 years at home and abroad were selected from GenBank for conservation analysis of antigenic epitopes. The antigenic epitopes were analyzed using Megalign of DNAstar software. 492 FPHEV 496 Conservation among ADV strains.

[0112] The results are as follows Figure 16 As shown, the antigen epitope has 100% homology with 24 strains of ADV published at home and abroad. 492 FPHEV 496 It is a good target for detecting the spread of ADV strains, which will help improve the specificity and universality of ADV detection technology, accelerate the purification of ADV, and lay the foundation for further prevention and control of the disease.

[0113] In summary, the hybridoma cell line ADV-GZ2104-H7 disclosed in the present invention has excellent biological properties. The monoclonal antibody prepared using it recognizes the antigenic epitope at amino acids 492 to 496 of the VP2 protein of ADV, and its polypeptide sequence is FPHEV. It has the characteristics of strong specificity, high affinity, strong recognition ability, good stability and high titer.

[0114] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A hybridoma cell line, whose deposit number is CGMCC No. 23875.

2. Use of the hybridoma cell line according to claim 1 in preparing a monoclonal antibody that recognizes the VP2 protein of mink Aleutian disease virus.

3. A monoclonal antibody, characterized in that The product is secreted and produced by the hybridoma cell line with the deposit number CGMCC No. 23875 as claimed in claim 1.

4. Use of the hybridoma cell line according to claim 1 or the monoclonal antibody according to claim 3 in the preparation of a product for detecting mink Aleutian disease virus.

5. The use according to claim 4, characterized in that The product is an ELISA kit, a colloidal gold test strip or an indirect immunofluorescence analysis kit.

6. A kit for detecting mink Aleutian disease virus, characterized in that: The kit contains an effective amount of the monoclonal antibody according to claim 3.

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