Hybridoma cell strain secreting anti-goose astrovirus cap protein monoclonal antibody, monoclonal antibody as well as preparation method and application of monoclonal antibody

Abstract

The invention discloses a hybridoma cell strain secreting an anti-goose astrovirus cap protein monoclonal antibody, the monoclonal antibody as well as a preparation method and application of the monoclonal antibody, and belongs to the technical field of immunity. According to the invention, a goose astrovirus cap protein is utilized to immunize a mouse, specific hybridoma cell strains 7B2 and 3D4 are separated and obtained from mouse ascites, and the monoclonal antibody is separated and purified. Further analysis shows that the epitope information recognized by the monoclonal antibody secreted by the hybridoma cell strain 7B2 is 153NTAGPESIDT162, homologous analysis is carried out on the epitope information in various virus strains, it is found that the epitope information is highly conserved in the goose astrovirus, it is indicated that the epitope information has the potential of specifically inhibiting the goose astrovirus, and the epitope information can be used for preparing the goose astrovirus. The method can be used for developing goose astrovirus treatment drugs or vaccines as a target spot.

Description

Technical Field

[0001] The invention relates to the field of immunotechnology, in particular to a hybridoma cell line secreting anti-goose astrovirus cap protein monoclonal antibody, the monoclonal antibody and a preparation method and application thereof. Background Art

[0002] Since 2017, an infectious disease with urate deposition as the main clinical manifestation has broken out in goose farms in my country. The death of a large number of goslings has caused serious losses to the goose farming industry. After the isolation and identification of the pathogen, the pathogen of this infectious disease is mainly goose astrovirus (GoAstV), which is called gout based on its clinical characteristics. It is common in goslings under 3 weeks old and has a high incidence rate. The mortality rate can be as high as 50%, causing huge economic losses to my country's goose farming industry. Although there are monoclonal antibodies against the cap protein of goose astrovirus, there are low detection efficiency and poor prevention and control effects. Therefore, there is still a lack of new antibodies for the diagnosis and prevention of goose astrovirus. Summary of the invention

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

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

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

[0006] The invention also provides a monoclonal antibody against goose astrovirus cap protein, wherein the monoclonal antibody is secreted and produced by the hybridoma cell line.

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

[0008] Preferably, the method comprises the step of utilizing the hybridoma cell line to secrete and produce the monoclonal antibody.

[0009] The present invention also provides an antigen epitope peptide recognized by the monoclonal antibody, wherein the amino acid sequence of the antigen epitope peptide is 153 NTAGPESIDT 162.

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

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

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

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

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

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

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

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 is the titer of polyclonal antibodies in the sera of immune mice;

[0019] Figure 2 These are the two positive hybridoma cells that can stably secrete. The left picture is 7B2, and the right picture is 3D4.

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

[0021] Figure 4 The results of monoclonal antibody WB identification; 7B2 and 3D4 are monoclonal antibodies 7B2 and 3D4, respectively, and POS is a positive control;

[0022] Figure 5 The results of the IEDB website’s prediction of GAstV-2 cap protein antigenic epitopes; the upper figure is the predicted map of antigenic epitopes, and the lower figure is the gene sequence of the predicted antigenic epitopes;

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

[0024] Figure 7 is the Western Blot screening result of the shortest linear antigen epitope of mAb 7B2; AE are the WB detection results of truncated proteins, 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 were connected to the pCold-MBP vector; M is a protein marker;

[0025] Figure 8 The sequence alignment result of the shortest linear antigen epitope of mAb 7B2 in related astroviruses; the black box is the shortest linear antigen epitope of mAb7B2. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Preparation and identification of goose astrovirus monoclonal antibodies

[0032] 1. Materials and methods

[0033] 1.1 Materials

[0034] Purified pCold I-cap protein (for the construction and purification methods of the recombinant protein, please refer to the document "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 colorimetric solution; HT culture medium additive (50×), HAT culture medium additive (50×).

[0035] 1.2 Methods

[0036] 1.2.1 Animal immunization

[0037] After the immunogen is identified, the concentration is determined. For the first immunization, Freund's complete adjuvant and 100μg / mouse protein are mixed in equal volumes, and the immunogen is emulsified with a vortex oscillator to form an oil-in-water structure. Take 6-8 week old BALB / c mice, disinfect the skin with alcohol, and inject the emulsified immunogen at multiple points on the back subcutaneously, and pay attention to the state of the mice. Immunize again after an interval of 14 days, use Freund's incomplete adjuvant and 100μg / mouse protein in equal volumes, mix them, and inject them subcutaneously on the back after emulsification. After 3 consecutive immunizations, blood is collected from the infraorbital sinus according to conventional methods, mouse serum is collected, and the mouse serum antibody titer is determined. When the titer reaches 1:10000 or above, fusion can be performed, and immunization is stopped. 3-4 days before fusion, impact immunization is performed, and 100μg / mouse (without adjuvant) of the immunogen is injected into the abdominal cavity. After 3-4 days, the spleen is taken for cell fusion.

[0038] 1.2.2ELISA 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, and incubate at 4°C overnight. Discard the coating buffer the next day and wash three times with PBST.

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

[0041] (3) Primary antibody incubation: Dilute the immune mouse serum from 1:200 to 1:409600 and add it to the reaction wells, 100 μL per well. At the same time, make a negative control well with the same dilution multiple. Incubate at 37°C for 1 h and wash three times with PBST.

[0042] (4) Secondary antibody incubation: 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 and incubate at room temperature in the dark for 10-15 min; add 50 μL of stop solution to each reaction well. Measure the OD of each reaction well with an ELISA reader within 10 min. 630 .

[0044] 1.2.3SP / 20 myeloma cell recovery

[0045] Take out SP / 20 myeloma cells from the liquid nitrogen tank and thaw them in a 37℃ water bath until the cells are completely thawed. 2Pour DMEM medium containing 10% fetal bovine serum into the cell culture flask, place it in a 37℃ CO2 constant temperature incubator, observe the cell status, and perform subculture 24h before cell fusion. Select cells with good growth and covering about 80% of the bottom, blow them down with DMEM and collect them into a centrifuge tube, wash them once and resuspend them for later use.

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

[0047] Take a BALB / c mouse for shock immunization, bleed it from the eye sockets and kill it. Collect the blood and centrifuge it to get the serum, which is the positive serum. Soak the mouse in 75% alcohol for 5 to 10 minutes for disinfection. Move the disinfected mouse into the clean bench and fix it on the dissection board. Fix the front and back limbs, cut the peritoneum, expose the spleen, take out the spleen and put it in a plate to wash twice, remove the fat tissue adhered to the outside of the spleen, and put it in a sterilized homogenizer. Add 5mL of DMEM basal culture medium to the homogenizer, grind, squeeze out the spleen cells until the spleen turns white, filter the cell suspension into a sterile 50mL centrifuge tube, add 10mL of culture medium to clean the lower homogenizer, and absorb the cell suspension and filter it into a 50mL centrifuge tube again. Centrifuge the liquid at 1000r / min for 10 minutes to remove the supernatant, and then resuspend it with DMEM basal culture medium for standby use. The whole process must be strictly aseptic. Feeder cells were obtained from the spleen of blank mice, and the method was the same as the preparation method of immune spleen cells. After centrifugation, they were resuspended in 50 mL of HAT medium and set aside.

[0048] 1.2.5 Cell fusion

[0049] (1) Blow off the cultured SP / 20 myeloma cells from the cell flask, wash twice with PBS, blow off with DMEM, count and resuspend, and adjust the cell concentration to 1 to 2×10 7 cells / mL. Mix with resuspended immune spleen cells at a ratio of 1:5-1:10 and centrifuge at 1000r / min for 10min. Gently remove the supernatant and invert the centrifuge tube on filter paper to absorb the remaining culture medium.

[0050] (2) Rotate the centrifuge tube along the graduated surface to shake and break up 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 containing SP / 20 myeloma cells and immune spleen cells in an insulated beaker filled with 37°C warm water, add 1 mL of preheated 50% PEG dropwise to the mixed cells within 1 min, stirring with a pipette tip while adding, continue stirring for 30 s, and let stand for 30 s.

[0052] (4) Then slowly add 10 mL of 37°C preheated DMEM medium as the stop solution. The specific method is: 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 time you add, add slowly and stir while adding. Centrifuge at 1000 rpm for 10 minutes, and then discard the supernatant.

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

[0054] 1.2.6 Screening of positive hybridoma cells

[0055] On the second day after cell fusion, observe for contamination. On the fourth day, when obvious cell colonies can be observed in the cell culture wells, perform a half-medium change, that is, aspirate 100μL of culture medium and replace it with 100μL HT medium. Continue to observe and perform the first full medium change when the cells grow to 1 / 4. Perform another full medium change the night before the antibody test. About 14 days after fusion, most cell culture wells show vigorous cell growth. When the cell culture wells are examined under a microscope, small grape-like clusters of hybridoma cells become more obvious and continue to grow. The cell supernatant is tested by indirect ELISA. The primary antibody is 100μL of cell supernatant. 100μL of 1:1000 diluted yin and yang serum is added to the positive and negative control wells. At OD 630 Read the numbers.

[0056] 1.2.7 Subcloning of positive hybridoma cells

[0057] The hybridoma cells identified as positive were subcloned by limiting dilution method as follows:

[0058] (1) Screening out positive hybridoma cells, preparing a cell suspension using HT medium and counting the cells;

[0059] (2) Diluting the hybridoma cells with HT medium to prepare cell suspensions at 100 cells / mL, 40 cells / mL, 20 cells / mL, and 10 cells / mL; adding the diluted cell suspensions to the feeder cells cultured the day before, respectively, to make the cell content of each well 5 cells / well, 2 cells / well, 1 cell / well, and 0.5 cells / well, and culturing in an incubator at 37° C. and 5% CO2;

[0060] (3) Observe the cell growth status every day. When only one colony is observed and grows to 1 / 3 to 1 / 2 of the plate, detect the antibody titer of the supernatant by indirect ELISA;

[0061] (4) After high-titer hybridoma cells are detected, subcloning and purification are continued for about 4 times until all cloned cells in the plate are single cell colonies and all are positive.

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

[0063] The culture method is basically the same as that of ordinary cells. The positive hybridoma cells of the above limiting dilution are transferred to a 24-well plate. When they grow to 1 / 3 to 1 / 2 of the plate, they are transferred to a 12-well plate. In this way, they are transferred to a 6-well plate until they are placed in a culture bottle. The freezing medium is prepared by mixing 90% fetal bovine serum and 10% DMSO. The supernatant medium is discarded, and the freezing solution is added and transferred to a cryopreservation tube, which is placed in a gradient cooling box and then placed in a -80°C refrigerator overnight, and then transferred to a liquid nitrogen tank. The recovery process of hybridoma cells is the same as that of ordinary cells.

[0064] 1.2.9 Indirect immunofluorescence analysis of monoclonal antibodies

[0065] The selected monoclonal antibodies were identified by indirect immunofluorescence (IFA) as follows:

[0066] (1) Inoculation: Chicken hepatoma cells (LMH) were inoculated into 96-well plates. When the cells grew to occupy 80%-90% of the wells and were in good condition, goose astrovirus was diluted 10, 100, or 1000 times and inoculated into the wells. At the same time, positive and negative controls were set up. After culturing for 24 hours, the culture medium was discarded and the cells were washed three times with PBS, each time for 3 minutes.

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

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

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

[0070] (5) Secondary antibody incubation: Add 100 μL of FITC fluorescently labeled secondary antibody (1:1000 dilution) to each well and incubate at 37°C for 1 h. Discard the secondary antibody and wash three times with PBST, each time for 3 min.

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

[0072] 1.2.10 Monoclonal Antibody WB Identification

[0073] The purified cap protein and virus were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with a 1% skim milk powder solution (prepared in PBS) in a 37°C incubator for 1 hour and washed three times with prepared PBST for 10 minutes each time. The primary antibody was the supernatant of the cells to be tested, incubated in a 37°C incubator for 1 hour, and then the PVDF membrane was washed three times with prepared PBST buffer for 10 minutes each time. The secondary antibody was a 1:2,000 diluted HRP-labeled goat anti-mouse IgG (diluted in PBST), and then the PVDF membrane was washed three times with prepared PBST buffer for 10 minutes each time, and finally observed with a DAB kit for color development.

[0074] 1.2.11 Preparation of ascites

[0075] BALB / c mice aged 6 to 10 weeks were selected and injected with 0.5 mL of Freund's incomplete adjuvant 5 to 7 days before cell injection for sensitization. Then, the hybridoma cells were diluted to 5 × 10 5 cells / mL, take 500μL of cell dilution and inject it into the abdominal cavity of mice after the abdomen is disinfected with alcohol, observe the mice every day until the abdominal cavity bulges into a circle, and then collect the ascites; after collection, place it in a centrifuge tube, then centrifuge it at 4℃5000rpm for 10min, and collect the ascites and store it at -80℃.

[0076] 2. Experimental results

[0077] 2.1 Titer of immunized mouse sera

[0078] After the mice were immunized with antigen three times, the titer of the mice was measured. The blood of the immunized mice was collected first, and then the serum was separated. The titer was measured by indirect ELISA, and negative serum was taken as a negative control. Figure 1 As shown, the results showed that the titers of the five immunized mice all reached above 1:51200, indicating that after three immunizations of mice with cap protein, the body induced the production of specific polyclonal antibodies against cap protein, and the spleen could be taken for cell fusion operation.

[0079] 2.2 Screening of positive hybridoma cells

[0080] About one to ten days after cell fusion, the culture medium can be seen to turn yellow. When the hybridoma cells grow to 1 / 3 to 1 / 2 of the bottom area, the cell supernatant is positively screened by indirect ELISA method, showing that 2 wells are positive. After several subcellular cloning, the experiment yielded 2 strains of positive hybridoma cells that can stably secrete. Figure 2 As shown, named 7B2, 3D4.

[0081] The above-mentioned positive hybridoma cell 7B2, named hybridoma cell GoAstV2 7B2, was deposited in the China Center for Type Culture Collection on November 10, 2024, with the deposit number CCTCC NO: C2024379, and the deposit address is 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, but no fluorescence signals were detected in LMH cells not infected with goose astrovirus (see Figure 3 ). The results showed that the screened monoclonal antibodies could recognize the natural virus.

[0084] 2.4 Western Blot Identification of Monoclonal Antibodies

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

[0086] Example 2 Identification of Antigenic Epitopes of Monoclonal Antibodies

[0087] 1. Experimental Materials

[0088] 1.1 Vectors, plasmids, and competent cells

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

[0090] 1.2 Main Reagents

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

[0092] Preparation of main reagents:

[0093] (1) Preparation of Western Blot Reagents:

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

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

[0096] c. PBST washing solution: Dissolve 500 μL Tween-20 in 1L PBS buffer and shake to mix.

[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] Clean bench (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), multifunctional molecular imaging system (Azure Bio, USA), constant temperature oscillator (Thermo, USA).

[0100] 2. Experimental methods

[0101] 2.1 Epitope prediction and primer design

[0102] The linear B cell antigen epitopes of the cap protein of GAstV-2 were predicted using the IEDB online website (http: / / www.iedb.org). According to the prediction results, the sequence was split into overlapping parts, and the overlapping part contained at least 8 amino acids. Primer 6 software was used for primer design, and XhoI and EcoRI restriction sites and protective bases were inserted into the upstream and downstream primers, which were sent to Sangon Biotech (Shanghai) Co., Ltd. for synthesis. The primer synthesis sequence is shown in Table 1.

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

[0104] 2.2 Construction of truncated recombinant plasmid

[0105] The synthesized primers were used for PCR amplification, and then the recombinant plasmid of the truncated gene was constructed through agarose gel recovery, restriction endonuclease digestion test, ligation to pCOLD-MBP expression vector, transformation, plasmid extraction, plasmid identification and other tests.

[0106] 2.3 Expression of truncated proteins

[0107] The constructed and successfully identified truncated recombinant plasmid was transformed into Rosetta competent medium for small-scale expression. The method was as follows: positive monoclonal colonies were picked and cultured in 2 mL of LB liquid medium containing ammonia. The reaction conditions were 37°C and 200×g. When the OD of the bacterial solution reached 600nm When the value reaches 0.6-0.8, add IPTG (1mM) at a ratio of 1:1000, 200×g, 16℃ for induction overnight; take 1mL of bacterial solution and centrifuge at 12000×g for 1min, discard the supernatant, resuspend the precipitate with 100μL PBS, add 25μL 5×Loading Buffer to the resuspended solution, boil at 100℃ for 10min, run SDS-PAGE protein gel (bacterial solution without IPTG was used as negative control). Western Blot analysis was performed using the prepared GAstV-2 monoclonal antibody as the primary antibody, with pCold I-cap protein as the positive control.

[0108] 2.4 Preliminary screening of antigenic epitopes

[0109] According to the prediction results, the pCOLD-MBP cap protein was first divided into multiple short peptides, and the monoclonal antibody was prepared as the primary antibody, and the pCold I-cap protein was used as the positive control for Western Blot analysis. According to the Western Blot results, the protein that reacts with the prepared monoclonal antibody was truncated and expressed until the expressed protein had a sequence of about 17 amino acids and then accurately located.

[0110] 2.5 Precise localization of antigenic epitopes

[0111] After multiple rounds of Western Blot analysis, the antigenic epitopes screened by preliminary positioning were reduced amino acids one by one to determine the shortest antigenic epitope. The gene sequence was determined based on the amino acid sequence, and the gene sequence was connected 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 a positive control to analyze the shortest linear antigenic epitope. The protein sequence to be synthesized by the recombinant plasmid is shown in Table 2, and the construction process was entrusted to Biosynthesis.

[0112] Table 2 Gene sequences of artificially synthesized recombinant plasmids

[0113] name Sequence number Amino acid sequence C3-1a SEQ ID NO: 1 <![CDATA[ 130 LVPLAGRANILGSVVFLDIEQEAN 153 ]]> C3-1b SEQ ID NO: 2 <![CDATA[ 149 EQEANTAGPESIDTICS 165 ]]> C3-1c SEQ ID NO: 3 <![CDATA[ 160 IDTIKARPHLELPIGSK 176 ]]> C3-1b-1 SEQ ID NO: 4 <![CDATA[ 150 QEANTAGPESIDTICS 165 ]]> C3-1b-2 SEQ ID NO: 5 <![CDATA[ 151 ANTAGPESIDTIKA 165 ]]> C3-1b-3 SEQ ID NO: 6 <![CDATA[ 152 ANTI-PESHITICS 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 NTAGPEDTI 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 ]]>

[0114] 2.6 Conservation analysis of the shortest antigenic epitope

[0115] After determining the shortest linear antigen epitope of mAb 7B2, in order to analyze its conservation, the present invention used MEGA 6.06 software to compare the corresponding regions of GAstV-2, GAstV-1, Human astrovirus-2, Duck astrovirus-2, Pocineastrovirus-2, Canine astrovirus, Turkey astrovirus-2 and Feline astrovirus-2 strains. The GenBank sequence numbers of the Cap genes of GAstV-2 strains are OL762473, OR234628.1, OR234615, QRN45266.1, UIW24841.1, UYR25377.1, UYR25380.1, WNG75970.1, OM455389, and MT934439; the GenBank sequence numbers of the Cap genes of GAstV-1 strains are MW353015, OL762472, OM264914, UIX56556.1, PP63758.1, and UYR25374.1; Human astrovirus-2, Duck astrovirus-2, Pocine astrovirus-2, Canine astrovirus, Turkey astrovirus-2, and Feline The GenBank sequence numbers of the Cap genes of astrovirus-2 strains are QKW80827.1, UNJ12754.1, AZB49326.1, WNA12464.1, NP_987088.1 and YP_008519303.1.

[0116] 3. Experimental results

[0117] 3.1 Epitope prediction and primer design

[0118] The GAstV-2 cap protein antigen epitope results predicted by the IEDB online website are as follows Figure 5 The present invention conducted 6 rounds of Western Blot analysis based on the prediction results to screen the shortest linear antigen epitope of mAb 7B2. The screening process is as follows: Figure 6 shown.

[0119] 3.2 Truncated expression of Cap protein and screening of the shortest linear antigen epitope of mAb 7B2

[0120] In order to verify the antigenic epitope of the above monoclonal antibody, a truncated recombinant plasmid was first constructed according to the predicted antigenic epitope, and the recombinant protein with a His tag was expressed in Escherichia coli. Figure 7As shown, the Cap protein was first 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, and 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 split into two truncated recombinant proteins, named C3-1 (130-176aa) and C3-2 (163-210aa), respectively, with corresponding protein sizes of approximately 49KDa, 48KDa and 49KDa. The monoclonal antibody only reacts with the C3-1 segment recombinant protein, but not with the C3-2 segment protein, so the antigenic epitope of the antibody should be located between 130-176aa. 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), and the corresponding recombinant protein sizes were approximately 47KDa and 47KDa, respectively. mAb 7B2 only reacts with the C3-1b (149-165aa) segment protein, and does not react with the C3-1a and C3-1c segment proteins, so the shortest antigen epitope should be located between 149-165aa. Next, the protein expression was carried out in a decreasing order of amino acids, and the artificially synthesized plasmids shown in Table 2 were expressed in Escherichia coli. First, mAb 7B2 was subjected to Western Blot analysis with six proteins, C3-1b-1, C3-1b-2, C3-1b-3, C3-1b-4, C3-1b-5, and C3-1b-6. The results showed that the antibody reacted strongly with C3-1b-1, C3-1b-2, C3-1b-3, and C3-1b-4, but weakly with C3-1b-5 and did not react with C3-1b-6. Therefore, the antigen epitope was located between 153 and 165aa. The C-terminal amino acids were further decreased for screening, and mAb 7B2 was found to react strongly with C3-1b-41, C3-1b-42, and C3-1b-43, but not with C3-1b-44 and C3-1b-45. Therefore, after multiple rounds of screening, the shortest antigen epitope of mAb 7B2 was determined to be 153-162aa, that is, the amino acid sequence is 153 NTAGPESIDT 162 , which was consistent with the predicted antigenic epitope.

[0121] 3.3 Conservation analysis of antigenic epitopes

[0122] To verify the conservation of the shortest linear antigen epitope of mAb 7B2, the gene sequences of five GAstV-2, two GAstV-1 and six other related astrovirus strains were compared using SnapGene software. 153 NTAGPESIDT 162 It is highly conserved in the GAstV-2 gene sequence and does not contain any mutation sites; it has a high homology with Turkeyastrovirus 2, but a low homology with other related astrovirus strains, less than 50% homology, such as Figure 8 As shown. Therefore, it can be proved that the shortest antigen epitope of mAb 7B2 is highly conserved and specific.

[0123] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all 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 deposit number of the hybridoma cell line is CCTCC NO: C2024379.

2. A monoclonal antibody against goose astrovirus cap protein, characterized in that: The monoclonal antibody is secreted and produced by the hybridoma cell line according to claim 1.

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

4. The method for preparing a monoclonal antibody according to claim 2 or 3, characterized in that: The method comprises the step of utilizing the hybridoma cell line according to claim 1 to secrete and produce the monoclonal antibody.

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

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

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

9. A reagent or kit for detecting goose astrovirus, characterized in that: Comprising the monoclonal antibody according to claim 2 or 3.

10. A drug or vaccine for preventing and treating goose astrovirus, characterized in that: Comprising the monoclonal antibody according to claim 2 or 3.

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