Hybridoma cell line secreting monoclonal antibody against goose astrovirus cap protein, monoclonal antibody, and preparation method and application thereof

Abstract

The present invention discloses a hybridoma cell line secreting monoclonal antibodies against goose astrovirus cap protein, the monoclonal antibodies, and their preparation methods and applications, belonging to the field of immunotechnology. In the present invention, mice are immunized with goose astrovirus cap protein, and specific hybridoma cell lines 7B2 and 3D4 are isolated from mouse ascites, and monoclonal antibodies are isolated and purified. Further analysis shows that the epitope information recognized by the monoclonal antibody secreted by hybridoma cell line 7B2 is 153 NTAGPESIDT 162 . Homology analysis of this epitope information is carried out among various virus strains, and it is found that this epitope information is highly conserved in goose astrovirus, indicating its potential to specifically inhibit goose astrovirus, and it can be used as a target for the development of therapeutic drugs or vaccines against goose astrovirus.

Description

Technical Field

[0001] The present invention relates to the field of immunology, and particularly to a hybridoma cell line secreting monoclonal antibodies against the cap protein of goose astrovirus, monoclonal antibodies, and their preparation methods and applications. Background Art

[0002] Through the isolation and identification of pathogens, the pathogen of this infectious disease is mainly Goose astrovirus (GoAstV), which is called gout disease according to its clinical characteristics. It mostly occurs in goslings within 3 weeks of age and has a relatively high incidence rate, with a maximum mortality rate of up to 50%. At present, although there are already monoclonal antibodies against the cap protein of goose astrovirus, there are situations of low detection efficiency and poor prevention and treatment effects. Therefore, there is still a lack of new antibodies for the diagnosis and prevention and treatment of goose astrovirus. Summary of the Invention

[0003] The purpose of the present invention is to provide a hybridoma cell line secreting monoclonal antibodies against the cap protein of goose astrovirus, monoclonal antibodies, and their preparation methods and applications, so as to solve the problems existing in the above-mentioned prior art and provide new antibodies for the diagnosis and prevention and treatment of goose astrovirus.

[0004] To achieve the above purpose, the present invention provides the following solutions:

[0005] The present invention provides a hybridoma cell line secreting monoclonal antibodies against the cap protein of goose astrovirus, named hybridoma cell GoAstV2 7B2. The preservation number of the hybridoma cell line is CCTCC NO: C2024379, the preservation time is November 10, 2024, and the preservation unit is the China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China.

[0006] The present invention also provides a monoclonal antibody against the cap protein of goose astrovirus, and the monoclonal antibody is secreted by the above-mentioned hybridoma cell line.

[0007] Preferably, the monoclonal antibody specifically binds to the cap protein antigen of goose astrovirus type 2.

[0008] Preferably, it includes the step of secreting the monoclonal antibody using the above-mentioned hybridoma cell line.

[0009] The present invention also provides an antigenic epitope peptide recognized by the above-mentioned monoclonal antibody, and the amino acid sequence of the antigenic epitope peptide is 153 NTAGPESIDT 162 .

[0010] The present invention also provides the use of the hybridoma cell line, or the monoclonal antibody, or the antigenic epitope peptide in the preparation of a reagent or kit for detecting goose astrovirus.

[0011] The present invention also provides the use of the hybridoma cell line, or the monoclonal antibody, or the antigenic epitope peptide in the preparation of a drug for preventing and treating goose astrovirus.

[0012] The present invention also provides the use of the hybridoma cell line, or the monoclonal antibody, or the antigenic epitope peptide in the preparation of a drug for preventing and treating diseases caused by goose astrovirus.

[0013] The present invention also provides a reagent or kit for detecting goose astrovirus, comprising the monoclonal antibody.

[0014] The present invention also provides a drug for preventing and treating goose astrovirus, comprising the monoclonal antibody.

[0015] The present invention discloses the following technical effects:

[0016] The present invention obtained two hybridoma cell lines 7B2 and 3D4 that secrete monoclonal antibodies against the cap protein of goose astrovirus, and the monoclonal antibodies secreted by the hybridoma cell lines can specifically recognize the cap protein of goose astrovirus. Further verification and analysis showed that the specific epitope peptide information recognized by the monoclonal antibody secreted by the hybridoma cell line 7B2 is 153 NTAGPESIDT 162 . The present invention performed a homology analysis on the epitope peptides recognized by the monoclonal antibodies secreted by 7B2 in different strains, and found that this epitope peptide information is highly conserved in goose astrovirus and has the potential to specifically inhibit the cap protein of goose astrovirus, and can be used as a target for the development of therapeutic drugs for goose astrovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0018] Figure 1 is the titer of the polyclonal antibody of the immune mouse serum;

[0019] Figure 2 are two positive hybridoma cells that can be stably secreted; among them, the left figure is 7B2 and the right figure is 3D4;

[0020] Figure 3Results of IFA identification for monoclonal antibodies; 7B2 and 3D4 are monoclonal antibodies 7B2 and 3D4 respectively, POS is the positive control, and NEG is the negative control;

[0021] Figure 4 Results of WB identification for monoclonal antibodies; 7B2 and 3D4 are monoclonal antibodies 7B2 and 3D4 respectively, and POS is the positive control;

[0022] Figure 5 Results of prediction of antigenic epitopes of GAstV-2 cap protein by the IEDB website; the upper figure is the prediction map of antigenic epitopes, and the lower figure is the gene sequence of antigenic epitope prediction;

[0023] Figure 6 Schematic diagram of the screening process for the shortest linear antigenic epitope of mAb 7B2;

[0024] Figure 7 Western Blot screening results for the shortest linear antigenic epitope of mAb 7B2; A-E are the WB detection results of truncated proteins respectively, 1: truncated pCold I-Cap protein; 2-20: C1(1-80aa), C2(70-140aa), C3(130-210aa), C3-1(130-176aa), C3-2(163-210aa), C3-1a(130-153aa), C3-1b(149-165aa), C3-1c(143-176aa), C3-1b-1(150-165aa), C3-1b-2(151-165aa), C3-1b-3(152-165aa), C3-1b-4(153-165aa), C3-1b-5(155-165aa), C3-1b-6(155-165aa), C3-1b-41(153-164aa), C3-1b-42(153-163aa), C3-1b-43(153-162aa), C3-1b-44(153-161aa), and C3-1b-45(153-160aa). These fragments are all ligated into the pCold-MBP vector; M is the protein Marker;

[0025] Figure 8 Sequence alignment results of the shortest linear antigenic epitope of mAb 7B2 in related astroviruses; the shortest linear antigenic epitope of mAb7B2 is within the black box. Detailed implementation manners

[0026] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0031] Example 1 Preparation and Identification of Monoclonal Antibodies Against Goose Astrovirus

[0032] 1. Materials and Methods

[0033] 1.1 Materials

[0034] Purified pCold I-cap protein (for the construction method and purification method of this recombinant protein, see the literature "Establishment of an Indirect ELISA Detection Method for Goose Astrovirus Capsid Protein"); Freund's complete adjuvant, Freund's incomplete adjuvant, LMH cells; Goose astrovirus; HRP-labeled goat anti-mouse IgG; FITC-labeled goat anti-mouse IgG; Myeloma cell line SP / 20; DAB-HRP chromogenic solution; HT medium additive (50×), HAT medium additive (50×).

[0035] 1.2 Methods

[0036] 1.2.1 Animal Immunization

[0037] After the immunogen is identified, its concentration is measured. For the first immunization, Freund's complete adjuvant and 100 μg / protein per mouse are mixed in equal volume, and the immunogen is emulsified with a vortex oscillator to form a water-in-oil structure. Take 6-8-week-old BALB / c mice, disinfect the skin with alcohol, and inject the emulsified immunogen subcutaneously at multiple points on the back, and observe the mouse status. Immunize again after 14 days, mix Freund's incomplete adjuvant and 100 μg / protein per mouse in equal volume, emulsify and inject subcutaneously on the back. After continuous immunization 3 times, collect mouse serum by orbital sinus blood collection according to the conventional method, and measure the antibody titer of mouse serum. When the titer reaches 1:10000 or above, fusion can be carried out, stop immunization, and give a boost immunization 3-4 days before fusion, inject 100 μg / mouse (without adjuvant) immunogen into the abdominal cavity, and take the spleen for cell fusion 3-4 days later.

[0038] 1.2.2 ELISA detection of polyclonal antibody titer

[0039] (1) Coating: Dilute the purified cap protein to 5 μg / mL with coating buffer, add 100 μL to each well, incubate overnight at 4 °C, discard the coating solution the next day, and wash three times with PBST.

[0040] (2) Blocking: After washing, block with 5% skim milk powder, add 300 μL to each well, incubate at 37 °C for 2 h, discard the blocking solution, and wash three times with PBST.

[0041] (3) Incubation with primary antibody: Dilute the immunized mouse serum from 1:200 to 1:409600, add 100 μL to the reaction wells, and at the same time make negative control wells with the same dilution factor, incubate at 37 °C for 1 h, and wash three times with PBST.

[0042] (4) Incubation with secondary antibody: Add 1:2000 HRP-labeled goat anti-mouse IgG secondary antibody, 100 μL per well, incubate at 37 °C for 1 h, and wash three times with PBST.

[0043] (5) Color development: Add 100 μL of TMB substrate solution to each reaction well for color development, protect from light at room temperature for 10-15 min; add 50 μL of stop solution to each reaction well. Measure the OD of each reaction well with an enzyme-linked immunosorbent assay reader within 10 min 630 .

[0044] 1.2.3 Resuscitation of SP / 20 myeloma cells

[0045] Take out SP / 20 myeloma cells from the liquid nitrogen tank, place them in a 37 °C water bath to thaw until the cells are completely melted, take the cells and put them into a 25 cm 2Pour DMEM medium containing 10% fetal bovine serum into a cell culture flask, place it in a 37°C CO2 incubator for culture, observe the cell state, and perform subculture 24 hours before cell fusion. Select cells with good growth and about 80% confluence at the bottom, pipette them down with DMEM and collect them into a centrifuge tube. After washing once, resuspend and set aside.

[0046] 1.2.4 Preparation of immune spleen cells and feeder cells

[0047] Take one BALB / c mouse that has been immunized by boosting, sacrifice it by orbital bleeding, collect the blood and centrifuge to obtain the serum, which is the positive serum. Soak the mouse in 75% alcohol for 5 to 10 minutes for disinfection, transfer the disinfected mouse into a laminar flow hood and fix it on a dissection board, fix the front and hind limbs, cut open the peritoneum, expose the spleen, take out the spleen, wash it twice in a petri dish, remove the adipose tissue adhering to the outside of the spleen, and put it into a sterilized homogenizer. Add 5 mL of DMEM basal medium to the homogenizer, grind it, squeeze out the spleen cells until the spleen turns white, filter the cell suspension into a sterile 50 mL centrifuge tube, add 10 mL of medium to wash the homogenizer, and aspirate the cell suspension and filter it again into a 50 mL centrifuge tube. Centrifuge the liquid at 1000 r / min for 10 minutes to remove the supernatant, and then resuspend it with DMEM basal medium, set aside. The whole process should be carried out under strict aseptic conditions. For feeder cells, take the spleen of a blank mouse, and the method is the same as that for the preparation of immune spleen cells. After centrifugation, resuspend it with 50 mL of HAT medium, set aside.

[0048] 1.2.5 Cell fusion

[0049] (1) Pipette the cultured SP / 20 myeloma cells from the cell flask, wash them 2 times with PBS, pipette them down with DMEM for counting and resuspension, and adjust the cell concentration to 1 to 2×10 7 cells / mL. Mix them with the resuspended immune spleen cells at a ratio of 1:5 - 1:10, and centrifuge at 1000 r / min for 10 min. Gently remove the supernatant, invert the centrifuge tube on a filter paper to adsorb the residual culture medium.

[0050] (2) Rotate the centrifuge tube along the graduated surface to disperse the cell pellet at the bottom of the tube, so that the cells are evenly spread on the bottom of the tube.

[0051] (3) Place the mixture of SP / 20 myeloma cells and immune spleen cells in a heat-insulated beaker containing warm water at 37°C, and gradually add 1 mL of pre-warmed 50% PEG dropwise to the mixed cells within 1 minute, stir with a pipette tip while adding, continue to stir for 30 s, and let it stand for 30 s.

[0052] (4) Then slowly add 10 mL of pre-warmed DMEM medium at 37 °C as the termination solution. The specific method is as follows: add 1 mL in the first minute, 2 mL in the second minute, 3 mL in the third minute, and 4 mL in the fourth minute. Each addition needs to be slow, and stir while adding. Centrifuge at 1000 rmp for 10 min, and then discard the supernatant.

[0053] (5) Add 50 mL of HAT medium to the centrifuge tube, and gently pipette the cells to resuspend and mix well. Then mix with the resuspended feeder cells, and pipette 200 μL per well onto a 96-well plate and culture in an incubator at 37 °C with 5% CO2.

[0054] 1.2.6 Screening of positive hybridoma cells

[0055] On the second day after cell fusion, check for contamination. By the fourth day, when obvious cell colonies can be observed in the cell culture wells, perform a semi-medium change, that is, aspirate 100 μL of the medium and replace it with 100 μL of HT medium, and continue to observe. When the cells grow to 1 / 4, perform the first full medium change, and then perform another full medium change the night before antibody detection. At about 14 days after fusion, most cell culture wells show vigorous cell growth. Check the cell culture wells under a microscope, and the hybridoma cell clusters like small grape clusters become more obvious and continue to grow. Detect the cell supernatant by indirect ELISA. The primary antibody is 100 μL of the cell supernatant, and add 100 μL of 1:1000 diluted positive and negative sera to the positive and negative control wells, and read the OD 630 value.

[0056] 1.2.7 Cell subcloning of positive hybridoma cells

[0057] Perform subcloning of the identified positive hybridoma cells by the limiting dilution method. The operation is as follows:

[0058] (1) Screen out the positive hybridoma cells, prepare a cell suspension with HT medium and perform cell counting;

[0059] (2) Dilute the hybridoma cells with HT medium to cell suspensions at 100 cells / mL, 40 cells / mL, 20 cells / mL, and 10 cells / mL; add the above-prepared cell suspensions to the feeder cells cultured the previous day respectively, so that the cell content per well is 5 cells / well, 2 cells / well, 1 cell / well, and 0.5 cells / well, and culture in an incubator at 37 °C with a 5% CO2 atmosphere;

[0060] (3) Observe the growth status of the cells every day. When observing a well with only one colony growing and growing to 1 / 3 to 1 / 2 of the plate, detect the antibody titer of the supernatant by indirect ELISA method;

[0061] (4) After detecting hybridoma cells with high titer, continue with about 4 rounds of subclonal purification until all the cloned cells in the plate are single cell colonies and all are positive.

[0062] 1.2.8 Expansion culture, cryopreservation and resuscitation of positive hybridoma cells

[0063] The culture method is basically the same as that of ordinary cells. Transfer the above-mentioned limited-diluted positive hybridoma cells to a 24-well plate. When they grow to 1 / 3 to 1 / 2 of the plate, transfer them to a 12-well plate, and then transfer them to a 6-well plate according to this method until they reach a culture flask; the cryopreservation medium is prepared by mixing 90% fetal bovine serum and 10% DMSO. Discard the supernatant medium, add the cryopreservation solution and transfer it to a cryotube. Place it in a gradient cooling box and then put it in a -80°C refrigerator overnight, and then transfer it to a liquid nitrogen tank; the resuscitation process of hybridoma cells is the same as that of ordinary cells.

[0064] 1.2.9 Indirect immunofluorescence analysis of monoclonal antibodies

[0065] Use indirect immunofluorescence (IFA) to identify and screen monoclonal antibodies. The steps are as follows:

[0066] (1) Virus inoculation: Inoculate chicken hepatoma cells (LMH) into 96 wells. When the cells grow to 80%-90% of the cell wells and the cell state is good, dilute goose astrovirus 10-fold, 100-fold, and 1000-fold, and inoculate it into the cell wells. At the same time, set up positive and negative controls. After culturing for 24 h, discard the medium and wash it 3 times with PBS, 3 min each time.

[0067] (2) Fixation: Add 100 μL of 75% ice ethanol to each well, place it in a 4°C refrigerator for fixation for 30 min - 1 h, discard the fixing solution, and wash it 3 times with PBS, 3 min each time.

[0068] (3) Blocking: Add 100 μL of 1% BSA blocking solution to each well, place it in a 37°C incubator for blocking for 1 h, discard the blocking solution, and wash it 3 times with PBST, 3 min each time.

[0069] (4) Primary antibody incubation: Add 100 μL of the supernatant of the cells to be tested and positive and negative controls to each well, place it in a 37°C incubator for incubation for 1 h, discard the primary antibody, and wash it 3 times with PBST, 3 min each time.

[0070] (5) Secondary antibody incubation: Add 100 μL of the secondary antibody labeled with FITC (diluted 1:1000) to each well, place it in a 37°C incubator for incubation for 1 h, discard the secondary antibody, and wash it 3 times with PBST, 3 min each time.

[0071] (6)Finally, add one drop of 50% glycerol to each well, observe under a fluorescence microscope, and take pictures for archiving.

[0072] 1.2.10 Identification of monoclonal antibody by WB

[0073] After separating the purified cap protein and virus by SDS-PAGE, transfer them to a PVDF membrane. Block the membrane with 1% skim milk solution (prepared with PBS) in an incubator at 37°C for 1 h, wash it 3 times with the prepared PBST, 10 min each time. The primary antibody is the supernatant of the cells to be tested, incubate it in an incubator at 37°C for 1 h, then wash the PVDF membrane 3 times with the prepared PBST buffer, 10 min each time; the secondary antibody is goat anti-mouse IgG labeled with HRP diluted 1:2000 (diluted with PBST), then wash the PVDF membrane 3 times with the prepared PBST buffer, 10 min each time, and finally develop and observe with a DAB kit.

[0074] 1.2.11 Preparation of ascites

[0075] Select BALB / c mice aged 6 - 10 weeks. 5 to 7 days before injecting the cells, sensitize them by injecting 0.5 mL of Freund's incomplete adjuvant in advance; then dilute the hybridoma cells with PBS to 5×10 5 cells / mL, take 500 μL of the cell dilution and inject it into the abdominal cavity of the mice whose abdomen has been disinfected with alcohol. Observe the mice every day until a round swelling appears in the abdominal cavity, then the ascites can be collected; place the collected ascites in a centrifuge tube, and then centrifuge at 5000 rpm at 4°C for 10 min. Store the collected ascites at -80°C.

[0076] 2. Experimental results [[ID=X]] [[ID=Y]]

[0077] 2.1 Titer of immune mouse serum

[0078] After immunizing the mice with the antigen three times, measure the titer of the mice. First, collect the blood of the immunized mice, then separate the serum, and use indirect ELISA to measure the titer. At the same time, take negative serum for negative control. As Figure 1 shown, the results show that the titers of the 5 immunized mice all reach above 1:51200, indicating that the cap protein induces the body to produce specific polyclonal antibodies against the cap protein after 3 immunizations of the mice, and the spleen can be taken for cell fusion operation.

[0079] 2.2 Screening of positive hybridoma cells

[0080] About one to ten days after cell fusion, it can be seen that the culture medium turns yellow. When the hybridoma cells grow to 1 / 3 - 1 / 2 of the bottom area, use the indirect ELISA method to screen the cell supernatant for positivity. It shows that 2 wells are positive. After several subcellular clones, the experiment obtains 2 positive hybridoma cells that can stably secrete, asFigure 2 As shown, named 7B2 and 3D4.

[0081] The above-mentioned positive hybridoma cell 7B2, named hybridoma cell GoAstV2 7B2, was deposited at the China Center for Type Culture Collection on November 10, 2024, with the deposit number CCTCC NO: C2024379 and the deposit address being Wuhan University, Wuhan, China.

[0082] 2.3 IFA identification of monoclonal antibodies

[0083] The monoclonal antibody was used for IFA detection of the virus. Obvious fluorescence signals were detected in the cytoplasm of LMH cells infected with goose astrovirus, and no fluorescence signals were detected in LMH cells not infected with goose astrovirus (see Figure 3 ). The results indicate that the screened monoclonal antibody can recognize the native virus.

[0084] 2.4 WB identification of monoclonal antibodies

[0085] The purified monoclonal antibody was diluted 1:500 as the primary antibody and used for Western Blot with the purified cap protein. The results showed (see Figure 4 ) that there was an obvious band at a relative molecular mass of 29 kDa. It indicates that the prepared monoclonal antibody against cap protein can react with the cap protein.

[0086] Example 2 Epitope identification of monoclonal antibodies

[0087] 1. Experimental materials

[0088] 1.1 Vectors, plasmids, competent cells

[0089] The pCOLD-MBP expression vector used in the present invention is stored in this laboratory and can also be obtained through regular commercial channels; the competent cell Rosetta was purchased from Beijing TransGen Biotech Co., Ltd.

[0090] 1.2 Main reagents

[0091] Premix Taq enzyme (TAKARA), Gel Extraction Kit (Beyotime, China), restriction endonucleases XhoI and EcoRI (TAKARA), T4 DNA ligase (NEB, USA), AxyPrep Plasmid Miniprep Kit (Axygen, USA), IPTG inducer (Beyotime, China), skim milk powder (BD, USA), His-tag antibody (Solarbio, China), goat anti-mouse IgG-HRP (Beyotime, China).

[0092] Preparation of main reagents:

[0093] (1) Preparation of Western Blot related reagents:

[0094] a. Blocking solution: Add 2 g of skim milk powder to 40 mL of PBST and mix well by shaking.

[0095] b. Phosphate buffer (pH 7.4): 0.2 g of potassium dihydrogen phosphate; 2.9 g of disodium hydrogen phosphate; 8 g of sodium chloride; Make up to 1 L with deionized water.

[0096] c. PBST washing solution: Dissolve 500 μL of Tween-20 in 1 L of PBS buffer and mix well by shaking.

[0097] (2) LB medium (pH 7.0): 10 g / L tryptone; 5 g / L yeast extract; 10 g / L sodium chloride.

[0098] 1.3 Main instruments and equipment

[0099] Laminar flow hood (Thermo, USA), ultra-high speed centrifuge (Beckman, USA), cell culture incubator (Thermo, USA), metal bath (Xinling, China), semi-dry transfer apparatus (Bio-Rad, USA), DYY-6C electrophoresis apparatus (Liuyi, China), multi-functional molecular imaging system (Azure Bio, USA), thermostatic oscillator (Thermo, USA).

[0100] 2. Experimental methods

[0101] 2.1 Prediction of antigenic epitopes and primer design

[0102] Use the IEDB online website (http: / / www.iedb.org) to predict the linear B-cell antigenic epitopes of the cap protein of GAstV-2. According to the prediction results, split the sequence with overlap, and the overlapping part should contain at least 8 amino acids. Use primer 6 software for primer design, insert XhoI and EcoRI restriction enzyme sites and protective bases into the upstream and downstream primers, and send them to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The primer synthesis sequences are shown in Table 1.

[0103] Table 1 Primer sequences corresponding to the expression of partial truncated proteins

[0104]

[0105] 2.2 Construction of truncated recombinant plasmids

[0106] Perform PCR amplification on the synthesized primers, and then construct recombinant plasmids of truncated genes through experiments such as agarose gel recovery, restriction enzyme digestion test, ligation to the pCOLD-MBP expression vector, transformation, plasmid extraction, and plasmid identification.

[0107] 2.3 Expression of truncated proteins

[0108] The constructed and successfully identified truncated recombinant plasmid was transformed into Rosetta competent cells for small-scale expression. The method is as follows: Pick a positive monoclonal colony and culture it in 2 mL of LB liquid medium containing ampicillin. The reaction conditions are 37 °C and 200 × g. When the OD 600nm value reaches 0.6 - 0.8, add IPTG (1 mM) at a ratio of 1:1000, and induce overnight at 200 × g and 16 °C. Pipette 1 mL of the bacterial solution and centrifuge at 12000 × g for 1 min. Discard the supernatant, resuspend the pellet with 100 μL of PBS, add 25 μL of 5× Loading Buffer to the resuspended solution, and boil and denature at 10--0 °C for 10 min. Run an SDS-PAGE protein gel (the bacterial solution without IPTG added is used as a negative control). Western Blot analysis was performed using the prepared GAstV-2 monoclonal antibody as the primary antibody, with the pCold I-cap protein as the positive control.

[0109] 2.4 Preliminary screening of antigenic epitopes

[0110] According to the prediction results, the pCOLD-MBP cap protein was first divided into multiple short peptides. Using the prepared monoclonal antibody as the primary antibody and the pCold I-cap protein as the positive control, Western Blot analysis was performed. According to the Western Blot results, the proteins that reacted with the prepared monoclonal antibody were continuously truncated and expressed until the expressed protein had about 17 amino acid sequences, and then precise localization was carried out.

[0111] 2.5 Precise localization of antigenic epitopes

[0112] After multiple rounds of Western Blot analysis, the antigenic epitopes preliminarily localized and screened were decreased by one amino acid each to determine the shortest antigenic epitope. According to the amino acid sequence, the gene sequence was determined, and the gene sequence was ligated to the pCOLD-MBP vector to construct a recombinant plasmid for expression. The expressed protein was subjected to Western Blot with the prepared monoclonal antibody, and the pCold I-cap protein was used as the positive control to analyze the shortest linear antigenic epitope. The protein sequences that need to be synthesized for the recombinant plasmid are shown in Table 2, and this construction process was entrusted to Sangon Biotech for synthesis.

[0113] Table 2 Gene sequences of artificially synthesized recombinant plasmids

[0114] Name Sequence number Amino acid sequence C3-1a SEQ ID NO: 1 <![CDATA 130 LVPLAGRANILGSVVFLDIEQEAN 153 > C3-1b SEQ ID NO: 2 <![CDATA 149 EQEANTAGPESIDTIKA 165 > C3-1c SEQ ID NO: 3 <![CDATA 160 IDTIKARPHLELPIGSK 176 > C3-1b-1 SEQ ID NO: 4 <![CDATA 150 QEANTAGPESIDTIKA 165 > C3-1b-2 SEQ ID NO: 5 <![CDATA 151 EANTAGPESIDTIKA 165 > C3-1b-3 SEQ ID NO: 6 <![CDATA 152 ANTAGPESIDTIKA 165 > C3-1b-4 SEQ ID NO: 7 <![CDATA 153 NTAGPESIDTIKA 165 > C3-1b-5 SEQ ID NO: 8 <![CDATA 154 TAGPESIDTIKA 165 > C3-1b-6 SEQ ID NO: 9 <![CDATA 155 AGPESIDTIKA 165 > C3-1b-41 SEQ ID NO: 10 <![CDATA 153 NTAGPESIDTIK 164 > C3-1b-42 SEQ ID NO: 11 <![CDATA 153 NTAGPESIDTI 163 > C3-1b-43 SEQ ID NO: 12 <![CDATA 153 NTAGPESIDT 162 > C3-1b-44 SEQ ID NO: 13 <![CDATA 153 NTAGPESID 161 > C3-1b-45 SEQ ID NO: 14 <![CDATA 153 NTAGPESI 160 >

[0115] 2.6 Analysis of the conservation of the shortest antigenic epitope

[0116] After determining the shortest linear epitope of mAb 7B2, in order to analyze its conservation, the present invention used MEGA6.06 software to compare the corresponding regions of GAstV-2, GAstV-1, Human astrovirus-2, Duck astrovirus-2, Pocine astrovirus-2, Canine astrovirus, Turkey astrovirus-2 and Feline astrovirus-2 strains. The GenBank sequence numbers of the Cap gene of the GAstV-2 strain are OL762473, OR234628.1, OR234615, QRN45266.1, UIW24841.1, UYR25377.1, UYR25380.1, WNG75970.1, OM455389, MT934439; the GenBank sequence numbers of the Cap gene of the GAstV-1 strain are MW353015, OL762472, OM264914, UIX56556.1, PP63758.1 and UYR25374.1; the GenBank sequence numbers of the Cap gene of Human astrovirus-2, Duck astrovirus-2, Pocine astrovirus-2, Canine astrovirus, Turkey astrovirus-2 and Feline astrovirus-2 strains are QKW80827.1, UNJ12754.1, AZB49326.1, WNA12464.1, NP_987088.1 and YP_008519303.1 respectively.

[0117] 3. Experimental results

[0118] 3.1 Prediction of epitope and primer design

[0119] The predicted results of the GAstV-2 cap protein epitope on the IEDB online website are as Figure 5 shown. The present invention performed a total of 6 rounds of Western Blot analysis on this prediction result to screen the shortest linear epitope of mAb 7B2, and the screening process is as Figure 6 shown.

[0120] 3.2 Truncated expression of Cap protein and screening of the shortest linear epitope of mAb 7B2

[0121] To verify the epitope of the above monoclonal antibody, first, a truncated recombinant plasmid was constructed according to the predicted epitope, and a recombinant protein with a His tag was expressed in Escherichia coli. As Figure 7As shown in the figure, for the first time, the Cap protein was divided into three truncated recombinant proteins, namely C1 (1-80aa), C2 (70-140aa), and C3 (130-210aa), with sizes of approximately 55KDa, 55KDa, and 49KDa respectively. The pCold I-cap protein was used as a positive control. The results showed that only mAb 7B2 reacted with the C3 segment protein, but not with the other two truncated proteins. Subsequently, the C3 protein was further divided into two truncated recombinant proteins, named C3-1 (130-176aa) and C3-2 (163-210aa), with corresponding protein sizes of approximately 49KDa, 48KDa, and 49KDa. This monoclonal antibody only reacted with the C3-1 segment recombinant protein and not with the C3-2 segment protein. Therefore, the antigenic epitope of this antibody should be between 130-176aa. For the third time, the C3-1 segment protein was divided into three overlapping short peptides, namely C3-1a (130-153aa), C3-1b (149-165aa), and C3-1c (143-176aa), with corresponding recombinant protein sizes of approximately 47KDa and 47KDa respectively. mAb 7B2 only reacted with the C3-1b (149-165aa) segment protein and not with the C3-1a and C3-1c segment proteins. Therefore, the shortest antigenic epitope should be located between 149-165aa. Next, protein expression with one-by-one amino acid reduction was carried out. According to the synthetic plasmids shown in Table 2, they were expressed in Escherichia coli. First, mAb 7B2 was subjected to Western Blot analysis with six proteins, namely C3-1b-1, C3-1b-2, C3-1b-3, C3-1b-4, C3-1b-5, and C3-1b-6. The results showed that this antibody reacted strongly with the four proteins C3-1b-1, C3-1b-2, C3-1b-3, and C3-1b-4, reacted weakly with the C3-1b-5 protein, and did not react with the C3-1b-6 segment protein. Therefore, the antigenic epitope was located between 153-165aa. Continuing to screen by reducing one amino acid at the C-terminus one by one, it was found that mAb 7B2 reacted strongly with the proteins C3-1b-41, C3-1b-42, and C3-1b-43 and did not react with the C3-1b-44 and C3-1b-45 segments. Therefore, after multiple rounds of screening, the shortest antigenic epitope of mAb 7B2 was determined to be 153-162aa, that is, the amino acid sequence was 153 NTAGPESIDT 162 , which was consistent with the predicted antigenic epitope.

[0122] 3.3 Conservative analysis of antigenic epitope

[0123] To verify the conservation of the shortest linear antigenic epitope of mAb 7B2, the gene sequences of 5 GAstV-2 strains, 2 GAstV-1 strains and 6 other related astrovirus strains were aligned by SnapGene software. The results showed that the epitope 153 NTAGPESIDT 162 was highly conserved in the GAstV-2 gene sequence and contained no mutation sites; it had a relatively high homology with Turkeyastrovirus 2, but a relatively low homology with other related astrovirus strains, less than 50% homology, as Figure 8 shown. Therefore, it can be proved that the shortest antigenic epitope of mAb 7B2 has high conservation and specificity.

[0124] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against goose astrovirus cap protein, characterized in that, The preservation number of the hybridoma cell line is CCTCC NO: C2024379.

2. A monoclonal antibody against the cap protein of goose astrovirus, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line described in claim 1.

3. The monoclonal antibody according to claim 1, wherein The monoclonal antibody specifically binds to the cap protein antigen of goose astrovirus type 2.

4. The method for preparing the monoclonal antibody according to claim 2 or 3, characterized in that, It includes the step of secreting the monoclonal antibody by using the hybridoma cell line described in claim 1.

5. An antigenic epitope peptide recognized by the monoclonal antibody according to claim 2 or 3, characterized in that The amino acid sequence of the antigenic epitope peptide is 153 NTAGPESIDT 162 .

6. Use of the hybridoma cell line according to claim 1, or the monoclonal antibody according to any one of claims 2-3, or the antigenic epitope peptide according to claim 5 in the preparation of a kit for detecting goose astrovirus.

7. Use of the hybridoma cell line according to claim 1, or the monoclonal antibody according to any one of claims 2-3, or the antigenic epitope peptide according to claim 5 in the preparation of a drug for preventing and treating goose astrovirus.

8. Use of the hybridoma cell line according to claim 1, or the monoclonal antibody according to any one of claims 2-3, or the antigenic epitope peptide according to claim 5 in the preparation of a drug for preventing and treating diseases caused by goose astrovirus.

9. A kit for detecting goose astrovirus, characterized in that, It includes the monoclonal antibody described in claim 2 or 3.

10. A drug for preventing and treating goose astrovirus, characterized in that, It includes the monoclonal antibody described in claim 2 or 3.

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