Goose astrovirus type 2-based hybridoma cell strain, construction method, monoclonal antibody and detection reagent

Abstract

The invention discloses a hybridoma cell strain based on type 2 goose astrovirus, a construction method, a monoclonal antibody and a detection reagent. The hybridoma cell strain is a hybridoma cell strain A10D12, the preservation number of the hybridoma cell strain is CCTCC (China Center for Type Culture Collection) NO: C202506, and the hybridoma cell strain is preserved in the China Center for Type Culture Collection on March 26, 2025; or the hybridoma cell strain is a hybridoma cell strain A12G1, the preservation number of the hybridoma cell strain A12G1 is CCTCC NO: C202507, and the hybridoma cell strain A12G1 is preserved in the China Center for Type Culture Collection on March 26, 2025. According to the invention, purified escherichia coli expressed recombinant 2-type goose astrovirus capsid protein is used for immunizing mice, two monoclonal antibodies specifically aiming at GAstV-2ORF2 protein are obtained, and B cell epitopes recognized by the monoclonal antibodies are identified. Based on the monoclonal antibodies, an efficient detection immunological detection method for the goose astrovirus type 2 is established.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to a hybridoma cell line based on goose astrovirus type 2, a construction method, a monoclonal antibody and a detection reagent. Background Art

[0002] Goose astrovirus (GAstV) belongs to the genus Avian Astrovirus and is a non-enveloped, single-stranded, positive-sense RNA virus of the family Astroviridae. The GAstV genome is approximately 7.2 nt long and consists of a 5' untranslated region (UTR), three open reading frames (ORF1a, ORF1b, and ORF2), and a 3' UTR. ORF1a and ORF1b encode nonstructural proteins involved in viral replication, while ORF2 encodes the sole capsid protein, which contains a relatively conserved N-terminal capsid core region (S and P1 domains) and a highly variable C-terminal spike domain (P2 domain). The capsid protein is closely associated with viral particle maturation and assembly, viral cellular tropism, viral pathogenicity, and antibody production. Based on nucleotide sequence differences in the ORF2 region, GAstV can be divided into two genotypes: GAstV-1 and GAstV-2. The ORF2 nucleotide sequence identity between GAstV-1 and GAstV-2 is only 40.4%–41.6%. Epidemiological survey data show that the main prevalent strain of goose astrovirus in my country is GAstV-2.

[0003] Goose Astrovirus Type 2, also known as the novel goose astrovirus, primarily infects goslings between 5 and 15 days old, causing gout characterized by urate deposits in the joints and visceral surfaces, and kidney enlargement and bleeding. Currently, there is no effective commercial vaccine to prevent and control GAstV-2 infection. Therefore, establishing efficient detection and diagnostic methods is crucial for the prevention and control of the disease.

[0004] At present, several nucleic acid detection-based methods have been developed for the detection of GAstV-2, including real-time PCR, loop-mediated isothermal amplification (LAMP), etc. However, these molecular detection methods are mainly suitable for laboratory diagnosis and can only be used to detect samples of acutely or subclinically infected goslings. In contrast, immunological methods are more suitable for the detection of antigens or antibodies in clinical settings and are more conducive to the comprehensive evaluation of the infection status of goslings. Generally speaking, the preparation and identification of highly sensitive and specific monoclonal antibodies are crucial for the establishment of serological detection methods. However, there is currently little research on monoclonal antibodies against type 2 goose astrovirus and the B cell epitopes they target. Summary of the Invention

[0005] In view of the above-mentioned prior art, the object of the present invention is to provide a hybridoma cell line, construction method, monoclonal antibody and detection reagent that can efficiently realize the serological detection of goose astrovirus type 2, with high sensitivity, high specificity and good reproducibility.

[0006] In order to achieve the above object, the present invention provides a method for constructing a hybridoma cell line based on goose astrovirus type 2, the construction method comprising:

[0007] S100, amplifying the genetic material of goose astrovirus type 2 using the amplification primers shown in SEQ ID No: 1 and SEQ ID No: 2 to obtain an amplified product;

[0008] S200, inserting the amplified product obtained in step S100 into an expression vector to construct a recombinant plasmid;

[0009] S300, transforming the recombinant plasmid obtained in step S200 into host cells and inducing expression to obtain ORF2 recombinant protein;

[0010] S400, using the ORF2 recombinant protein obtained in step S300 to immunize animals, taking spleen cells from the immunized animals and fusing them with myeloma cells, followed by subcloning and screening to obtain a hybridoma cell line based on goose astrovirus type 2.

[0011] Preferably, the expression vector in step S200 is a prokaryotic expression vector pET-30a; and / or,

[0012] The host cell in step S300 is selected from Escherichia coli BL21 cells; and / or,

[0013] The animals in step S400 are selected from BALB / c mice.

[0014] The present invention also provides a hybridoma cell line obtained by the construction method described above, wherein the hybridoma cell line is hybridoma cell line A10D12 (Hybridoma cell line A10D12), whose preservation number is CCTCC NO: C202506, and it was deposited in the China Center for Type Culture Collection on March 26, 2025.

[0015] The present invention also provides a hybridoma cell line obtained by the above-described construction method, wherein the hybridoma cell line is a hybridoma cell line A12G1 strain, whose preservation number is CCTCC NO: C202507, and was deposited in the China Center for Type Culture Collection on March 26, 2025.

[0016] The present invention also provides a monoclonal antibody against the ORF2 protein of goose astrovirus type 2, which is prepared from the hybridoma cell line described above. That is, the hybridoma cell line provided by the present invention can be used to prepare a monoclonal antibody against the ORF2 protein of goose astrovirus type 2.

[0017] Preferably, the heavy chain of the monoclonal antibody against type 2 goose astrovirus ORF2 protein is of IgG1 type, and the light chain is of κ type.

[0018] Preferably, the antigenic epitope of the type 2 goose astrovirus ORF2 protein specifically recognized by the monoclonal antibody against the type 2 goose astrovirus ORF2 protein prepared by the hybridoma cell line A10D12 is 36 YKPQKLPMKA 45 .

[0019] Preferably, the antigenic epitope of the type 2 goose Astrovirus ORF2 protein specifically recognized by the monoclonal antibody against the type 2 goose Astrovirus ORF2 protein prepared by the hybridoma cell line A12G1 is 1MADRA5.

[0020] The present invention also provides a detection reagent for goose astrovirus type 2, wherein the detection reagent comprises the hybridoma cell line described above, or comprises the monoclonal antibody against goose astrovirus type 2 ORF2 protein described above.

[0021] Preferably, the detection reagent is an indirect competitive ELISA detection reagent.

[0022] Preferably, the detection reagent uses the monoclonal antibody against the type 2 goose astrovirus ORF2 protein as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG as the secondary antibody.

[0023] Through the above technical solution, the present invention used purified recombinant goose astrovirus type 2 capsid protein expressed in Escherichia coli to immunize mice, obtaining two monoclonal antibodies specific for the GAstV-2 ORF2 protein and identifying the B cell epitopes recognized by these monoclonal antibodies. Based on these monoclonal antibodies, an efficient immunological detection method for goose astrovirus type 2 was established. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0025] Figure 1A This is a diagram showing the results of SDS-PAGE analysis of the ORF2 recombinant protein in Detection Example 1;

[0026] Figure 1B This is the result of Western blotting detection of purified ORF2 recombinant protein using anti-His tag antibody in Detection Example 1;

[0027] Figure 1CThis is a graph showing the results of Western blotting detection of purified ORF2 recombinant protein using serum from mice immunized with purified ORF2 protein in Detection Example 1;

[0028] Figure 2A This is the result of IFA identification of LMH cells using monoclonal antibodies in detection example 2;

[0029] Figure 2B This is a graph showing the results of Western blotting of LMH cells using a monoclonal antibody in Detection Example 2;

[0030] Figure 2C This is the result of immunohistochemical detection of A10D12 monoclonal antibody and A12G1 monoclonal antibody in detection example 2;

[0031] Figure 3A is a schematic diagram of the ORF2 protein domain of goose astrovirus type 2 in detection example 3;

[0032] Figure 3B This is the IFA test result of 293 cells transfected with different recombinant plasmids in Test Example 3;

[0033] Figure 4A This is an animated diagram of the B cell epitope analysis in ORF2 of the GAstV-2YC20 structural protein recognized by the A10D12 monoclonal antibody and the A12G1 monoclonal antibody in Detection Example 3;

[0034] Figure 4B This is a spheroid model diagram for the B cell epitope analysis in ORF2 of the GAstV-2YC20 structural protein recognized by the A10D12 monoclonal antibody and the A12G1 monoclonal antibody in Detection Example 3;

[0035] Figure 4C is a graph showing the comparison of the amino acid sequences of GAstV-2YC20 and other astrovirus structural proteins in detection example 3;

[0036] Figure 5A This is the test result diagram of the sensitivity test in the verification case;

[0037] Figure 5B This is a graph showing the detection results of the specificity test in the verification example. DETAILED DESCRIPTION

[0038] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0039] The technical solutions of the present invention are described in detail below through specific examples. The GAstV-2YC20 strain of goose astrovirus type 2 was isolated and preserved by the Jiangsu Provincial Key Laboratory of Veterinary Biopharmaceutical High-Tech Research; the prokaryotic expression vector pET-30a was purchased from Sigma-Aldrich; Escherichia coli BL21 cells were commercially available from Nanjing Novozymes Biotechnology Co., Ltd.; BALB / c mice were purchased from Huachuang Zhongnuo Co., Ltd. in Taizhou, China; SP2 / 0 cells were derived from a shared cell line in the laboratory of Jiangsu Agricultural and Animal Husbandry Science and Technology Vocational College; and one-day-old healthy goose chicks were purchased from Jiangsu Jinpeng Company.

[0040] The His tag antibody was a commercially available product from Proteintech, Wuhan, China; the RNA extraction kit was the commercially available MiniBEST Universal RNA Extraction Kit (TaKaRa) from Takara Biotech (Dalian) Co., Ltd.; the One-step RT-PCR Kit was the commercially available Titanium One-Step RT-PCR Kit (TaKaRa) from Takara Biotech (Dalian) Co., Ltd.; Sal I restriction endonuclease, Xho I restriction endonuclease, and T4 DNA ligase were commercially available products (TaKaRa) from Takara Biotech (Dalian) Co., Ltd.; IPTG was a commercially available product from Jiangsu Kangwei Century Biotechnology Co., Ltd.; Ni-NTA agarose affinity resin and BCA protein concentration assay kit were commercially available products from Beyotime; Freund's complete adjuvant was a commercially available product from Sigma-Aldrich; PEG1500 was a commercially available product from Roche; and the typing ELISA kit was a commercially available product from Thermo Fisher Scientific, Inc. The FITC-labeled goat anti-duck IgG was a commercial product of KPL; the HRP-labeled goat anti-mouse IgG was a commercial product of KPL.

[0041] Among them, in the present invention, the operation method of Western blotting identification is as follows:

[0042] After the sample to be identified was separated by 12% SDS-PAGE, it was transferred to a polyvinylidene fluoride (PVDF) membrane and blocked with 5% skim milk powder for 1 hour at room temperature. Subsequently, the membrane was incubated at 4°C overnight and a His tag antibody diluted 1:1000 was used, or a monoclonal antibody in TBS blocking solution containing 1% skim milk powder was used as the primary antibody (specifically, a His tag antibody diluted 1:1000 was used as the primary antibody for Western blotting detection of ORF2 recombinant protein in Detection Example 1, and a monoclonal antibody in TBS blocking solution containing 1% skim milk powder was used as the primary antibody for Western blotting detection of monoclonal antibodies in Detection Example 2). After washing, the membrane was incubated with a horseradish peroxidase-labeled goat anti-mouse IgG antibody (KPL) diluted 1:20,000 as the secondary antibody in blocking solution. Finally, the protein bands were visualized by chemiluminescence using an ECL kit (NCM Biotech).

[0043] Regarding IFA testing, the specific operation method is as follows:

[0044] LMH cells infected with the GAstV-2YC20 strain or transfected with ORF2 and its truncated fragments were fixed with a pre-chilled acetone-ethanol solution (3:2) for 5 minutes. Subsequently, they were blocked with 3% BSA and incubated with diluted monoclonal antibodies at 37°C for 1 hour. After washing with PBS, fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (KPL) was added as a secondary antibody. Finally, the stained cells were observed using an inverted fluorescence microscope.

[0045] Regarding IHC (immunohistochemistry), the specific operation methods are as follows:

[0046] One-day-old healthy goose chicks were orally inoculated with 0.3 mL of GAstV-2YC20 strain (0.3 × 10 3.75 TCID 50 Each goose received an oral dose of the same volume of PBS. Five days after injection, the geese were euthanized with an intravenous injection of sodium pentobarbital, and their kidneys and livers were collected and fixed in 4% paraformaldehyde. For immunohistochemistry, monoclonal antibodies (mAbs) were used as primary antibodies, and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (KPL) was used as a secondary antibody.

[0047] The monoclonal antibodies mentioned in the above Western blotting, IFA and IHC tests are all monoclonal antibodies against the ORF2 protein of goose astrovirus type 2 prepared in Preparation Example 3 of the present invention.

[0048] Preparation Example 1: Expression and purification of ORF2 recombinant protein

[0049] RNA of GAstV-2YC20 strain type 2 goose astrovirus was extracted using an RNA extraction kit. One-step RT-PCR Kit and SEQ ID No: 1 (ORF2-F: 5'-TAAGAAGGAGATATACATATGGCAGACAGGGCGGTGGC-3') and SEQ ID No: 2 (ORF2-R:

[0050] The extracted RNA was amplified using the primers shown in Figure 5'-GCTTCCTTTCGGGCTTTGTCACTCATGTCCGCCCTTCTC-3' to obtain an amplified product. The purified amplified product fragment was inserted into the prokaryotic expression vector pET-30a by homologous recombination to obtain the recombinant plasmid pET-ORF2.

[0051] The resulting recombinant plasmid pET-ORF2 was transformed into Escherichia coli BL21 cells and induced for expression with 0.4 mmol / L IPTG at 37°C for 4 hours to obtain the ORF2 recombinant protein. The resulting ORF2 recombinant protein was purified using Ni-NTA agarose affinity resin. The protein concentration of the purified ORF2 recombinant protein was determined using a BCA protein concentration assay kit, resulting in a protein concentration of 160 ng / μL.

[0052] Preparation Example 2: Construction of a hybridoma cell line for preparing monoclonal antibodies against goose astrovirus type 2 ORF2 protein

[0053] 50 μg of the purified ORF2 recombinant protein obtained in Preparation Example 1 was mixed with 50 μg of Freund's complete adjuvant and used to immunize 6-week-old female BALB / c mice. Subsequently, the female BALB / c mice were boosted every two weeks with the same dose of the aforementioned reagent (i.e., 50 μg of purified ORF2 protein mixed with 50 μg of Freund's complete adjuvant). Three days after the fourth booster immunization, spleen cells from the immunized female BALB / c mice were collected. Unimmunized 6-week-old female BALB / c mice were used as negative controls. After testing, the specific antibody titer of the 6-week-old female BALB / c mice after the above immunization reached no less than 1:64,000, confirming that they can be used for subsequent cell fusion operations.

[0054] Splenocytes from the immunized female BALB / c mice obtained above were fused with SP2 / 0 cells using PEG1500. The fused cells were screened by indirect ELISA (iELISA) using the ORF2 recombinant protein obtained in Example 1 as the coating antigen, yielding two positive hybridoma cell lines. Three rounds of subcloning of these two hybridoma cell lines resulted in the successful establishment of two hybridoma cell lines stably expressing monoclonal antibodies against the ORF2 protein of goose astrovirus type 2. These hybridoma cell lines were designated hybridoma cell line A10D12 and hybridoma cell line A12G1, respectively.

[0055] Preparation Example 3: Preparation of monoclonal antibodies against ORF2 protein of goose astrovirus type 2

[0056] The method for preparing monoclonal antibodies against the ORF2 protein of goose astrovirus type 2 is as follows:

[0057] Healthy female BALB / c mice aged 6-8 weeks were selected and pretreated by intraperitoneal injection of 0.5 mL of pristane under sterile conditions to induce the formation of an inflammatory microenvironment in the peritoneal cavity. After 7-14 days, hybridoma cells (≥5×10 6 cells / mL, with viability >90% as confirmed by trypan blue staining) were resuspended in sterile PBS and inoculated into pretreated mice (5×10 5 -1×10 6 After inoculation, the animals were continuously monitored. When the abdominal cavity was significantly distended, sterile peritoneal puncture was performed using an 18G needle under light anesthesia, and ascites was collected in batches. The collected ascites was centrifuged at 2000×g for 10 minutes to remove cellular debris. The fluid was then sterilized through a 0.22μm filter and purified by Protein A / G affinity chromatography to obtain highly pure (>90%) monoclonal antibodies.

[0058] Among them, the monoclonal antibody produced by hybridoma cell line A10D12 is recorded as A10D12 monoclonal antibody; the monoclonal antibody produced by hybridoma cell line A12G1 is recorded as A12G1 monoclonal antibody.

[0059] Detection Example 1: Identification of ORF2 recombinant protein

[0060] The expression of the purified ORF2 recombinant protein in Preparation Example 1 was identified by SDS-PAGE and Western blotting (the primary antibody used in Western blotting identification was a His-tag antibody). The identification results are as follows: Figure 1A-Figure 1C shown. Specifically:

[0061] Figure 1A: is the SDS-PAGE analysis result of ORF2 recombinant protein; wherein, band M is a molecular weight marker, band 1 is the bacterial liquid after uninduced culture transformed with pET-ORF2, band 2 is the bacterial liquid after induced culture transformed with pET-ORF2; band 3 is the purified ORF2 recombinant protein in Preparation Example 1.

[0062] Figure 1B The figure shows the results of Western blotting detection of purified ORF2 recombinant protein using anti-His tag antibody (i.e., His-tag monoclonal antibody); Figure 1C The figure shows the results of Western blotting detection of the purified ORF2 recombinant protein obtained in Preparation Example 1 using the mouse serum (i.e., mouse polyclonal antibody) immunized with the purified ORF2 recombinant protein. Figure 1B and Figure 1C In the figure, band M is a molecular weight marker, and band 1 is the ORF2 recombinant protein purified in Preparation Example 1.

[0063] like Figure 1A As shown in FIG, the molecular weight of the purified ORF2 recombinant protein obtained in Preparation Example 1 was approximately 90 kDa, which was in line with expectations; Figure 1B and Figure 1C As shown, the purified ORF2 recombinant protein obtained in Preparation Example 1 can be recognized by anti-His tag antibodies and serum of mice immunized with the purified ORF2 protein, verifying the successful expression of the ORF2 recombinant protein obtained in Preparation Example 1.

[0064] Test Example 2: Identification of Monoclonal Antibodies Targeting ORF2 Protein of Goose Astrovirus Type 2

[0065] The monoclonal antibodies prepared in Preparation Example 3 were identified using a typing ELISA kit. The identification showed that the heavy chains of the monoclonal antibodies produced by hybridoma cell lines A10D12 and A12G1 were both IgG1 type, and the light chains were κ type.

[0066] The monoclonal antibodies produced by hybridoma cell lines A10D12 and A12G1 were tested by IFA and Western blotting respectively to identify LMH cells infected with GAstV-2YC20 strain. The results were as follows: Figure 2A and Figure 2B As shown. Among them, Figure 2A The results of IFA identification of LMH cells using monoclonal antibodies are shown in Figure 1. Figure 2AThe three middle figures, from left to right, respectively, show the monoclonal antibody produced by hybridoma cell line A10D12 (i.e., A10D12 monoclonal antibody) detecting LMH cells infected with GAstV-2YC20 strain, the monoclonal antibody produced by hybridoma cell line A12G1 (i.e., A12G1 monoclonal antibody) detecting LMH cells infected with GAstV-2YC20 strain, and a negative control; Figure 2B The figure shows the results of Western blotting detection of LMH cells using monoclonal antibodies. Figure 2B The three middle pictures from left to right are A10D12 monoclonal antibody detecting LMH cells infected with GAstV-2YC20 strain, A12G1 monoclonal antibody detecting LMH cells infected with GAstV-2YC20 strain, and negative control.

[0067] A10D12 monoclonal antibody and A12G1 monoclonal antibody were used for immunohistochemistry (IHC) detection, and the results were as follows Figure 2C As shown, Figure 2C The three pictures in the middle correspond from left to right to the infected goslings with A10D12 monoclonal antibody as the primary antibody, the infected goslings with A12G1 monoclonal antibody as the primary antibody, and the uninfected goslings. IHC results showed that A10D12 monoclonal antibody and A12G1 monoclonal antibody can specifically recognize GAstV-2 in the kidneys and livers of infected goslings. Figure 2C ), while no signal was detected in the control group.

[0068] Figure 2A 、 Figure 2B The negative controls mentioned in the test were all tested on LMH cells that were not infected with GAstV-2YC20 strain. Figure 2C The control group mentioned in the experiment was the kidney and liver of goose chicks that were not infected with GAstV-2YC20 strain.

[0069] The above test results show that both A10D12 monoclonal antibody and A12G1 monoclonal antibody have strong specific recognition ability for type 2 goose astrovirus ORF2 protein.

[0070] Detection Example 3: B cell epitope localization and analysis

[0071] According to the three domains of the ORF2 gene of goose astrovirus type 2, i.e. S, P1 and P2, three fragments of the GAstV-2YC20 ORF2 gene were first amplified, and then the N-terminus of the S gene fragment was truncated to varying degrees to obtain S gene fragments of different lengths. The primers for amplifying the ORF2 gene with different deletions are listed in Table 1. The amplified gene fragments of different lengths were cloned into the vector pSC-Flag using a kit by a one-step cloning method to obtain a series of recombinants containing ORF gene fragments of different lengths. After correct identification, the obtained recombinant plasmid was transfected into 293 cells. The next day after transfection, the monoclonal antibody to be tested (i.e., the A10D12 monoclonal antibody and the A12G1 monoclonal antibody prepared in Preparation Example 3) was used as a primary antibody, and FITC-labeled goat anti-mouse antibody was used as a secondary antibody, and an indirect immunofluorescence assay (IFA) was performed according to a conventional method.

[0072] Table 1

[0073]

[0074]

[0075] Schematic diagram of the ORF2 protein domain of goose astrovirus type 2 Figure 3A As shown in the figure, the IFA test results of 293 cells after transfection with different recombinant plasmids are shown in the figure Figure 3B As shown. Among them, Figure 3B In the figure, the upper mark refers to the marker of the ORF2 gene fragment contained in the recombinant plasmid used to transfect 293 cells. The marks on the left represent, from top to bottom, the positive control, A10D12 monoclonal antibody as the primary antibody, and A12G1 monoclonal antibody as the primary antibody.

[0076] pass Figure 3A and Figure 3B It can be seen that both A10D12 and A12G1 react with 293 cells transfected with S gene fragments, but do not react with 293 cells transfected with P1 or P2 gene fragments, indicating that the epitopes recognized by A10D12 and A12G1 are both located in the S domain. In addition, A12G1 recognizes S-255 but not S-250, and A10D12 recognizes S-220 but not S-210, indicating that the B cell epitopes recognized by A12G1 and A10D12 are located in 1MADRA5 and 1MADRA6 of the S domain of ORF2 protein, respectively. 36 YKPQKLPMKA 45 .

[0077] The 3D structure of GAstV-2YC20 structural protein ORF2 was predicted using the online analysis tool Phyre, and the B cell epitopes recognized by the monoclonal antibodies were visualized using the software PyMOL. Figure 4A and Figure 4B As shown (specifically, the three-dimensional structural model diagram of GAstV-2YC20 ORF2 structural protein generated by PyMOL, Figure 4A For animated pictures, Figure 4B (Figure 2 is a sphere model diagram). It can be seen that the epitopes recognized by A10D12 and A12G1 monoclonal antibodies are both located on the surface of the ORF2 structural protein. Among them, the epitope 1MADRA5 recognized by A12G1 monoclonal antibody forms a random coil secondary structure, and the epitope recognized by A10D12 monoclonal antibody 36 YKPQKLPMKA 45 Partially forms an α-helical secondary structure.

[0078] To analyze the conservation of the two B cell epitopes of GAstV-2YC20 ORF2 structural protein between GAstV-2 and other astrovirus strains, the amino acid sequences of GAstV-2YC20 and other astrovirus structural proteins were further aligned and analyzed using Lasergene MegAlign software. Figure 4C As shown, specifically, the epitopes recognized by A12G1 monoclonal antibody and A10D12 monoclonal antibody are aligned with the corresponding regions of different avian influenza virus strains including: GAstV-2, GAstV-1, ANV-1, ANV-2, DAstV-1, DAstV-2, DAstV-3, DAstV-4, TAstV-1, TAstV-2 and CAstV-A. The red boxes in the figure represent different epitopes of ORF2 protein. Figure 4C The comparison results showed that the B cell epitope 1MADRA5 of GAstV-2YC20 recognized by A12G1 monoclonal antibody was completely consistent with other GAstV-2 strains, but had 2 to 5 amino acid substitutions with other astrovirus strains. 36 YKPQKLPMKA 45 The two B cell epitopes recognized by the A10D12 and A12G1 monoclonal antibodies are highly conserved across GAstV-2 strains, with only a single amino acid mutation found in strains GTF07 and GTF04, respectively, while they share only 2-3 amino acids with other astrovirus strains. These results indicate that the two B cell epitopes recognized by the A10D12 and A12G1 monoclonal antibodies are highly conserved across GAstV-2 strains but differ significantly from those with other astrovirus strains.

[0079] Example 1. Establishment and optimization of indirect competitive ELISA (icELISA) method

[0080] The purified ORF2 recombinant protein from Preparation Example 1 was serially diluted with sodium carbonate buffer to various concentrations (0.125 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1.0 μg / mL, and 2.0 μg / mL for a single variable multi-step gradient experiment). The plate was then coated overnight at 4°C on an ELISA plate, with triplicate wells for each concentration. The plate was washed five times with 200 μL of PBST, blocked with 100 μL of 5% skim milk powder at 37°C for 30 min-2 h, and then washed five times with PBST. Add 50 μL of GAstV-2 positive serum (i.e., serum of goslings infected with GAstV-2) or negative serum (i.e., serum of goslings not infected with GAstV-2) diluted with blocking buffer (blocking buffer ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512 for single variable multi-gradient experiments) and 50 μL of A10D12 monoclonal antibody diluted with blocking buffer (blocking buffer ratios of 1:1250, 1:2500, 1:5000, 1:10000, 1:20000, and 1:40000 for single variable multi-gradient experiments) to each well and incubate at 37°C for 1-2 hours. After washing five times with PBST, add 100 μL of HRP-conjugated goat anti-mouse IgG as a secondary antibody to each well (using blocking buffer at dilutions of 1:5000, 1:10,000, 1:20,000, and 1:40,000 for single-variable multi-step gradient experiments). Incubate at 37°C for 1 hour and then wash five times with PBST. Add 100 μL of colorimetric solution to each well and incubate at 37°C in the dark for 5-30 minutes. Terminate the reaction by adding 50 μL of 2 mol / L sulfuric acid. Read the OD value at 450 nm using a plate reader. Optimal reaction parameters are optimized based on the maximum negative / positive (N / P) ratio.

[0081] The optimal reaction parameters finally determined after optimization were: coating with 0.5 μg / mL of the purified ORF2 recombinant protein in Preparation Example 1, incubation in a volume of 100 μL at 37°C for 1 hour; blocking with 5% skim milk powder at 37°C for 30 minutes; serum samples were diluted with blocking solution at a ratio of 1:16, and monoclonal antibody A10D12 was diluted with blocking solution at a ratio of 1:10000; competition reaction was carried out at 37°C for 2 hours; horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody was diluted at 1:10000; reaction with TMB substrate (i.e., color development solution) at 37°C for 15 minutes; and the reaction was terminated by adding 50 μL of 2 mol / L sulfuric acid.

[0082] Example 2: Determination of critical value of indirect competitive ELISA method

[0083] Utilize the icELISA condition optimized in Example 1 to detect 30 parts of GAstV-2 clinical negative serum, measure its absorbance value when 450nm, and calculate inhibition rate (PI) according to the formula. Calculate the average inhibition rate (X) and standard deviation (SD) of 30 parts of negative serum, and determine the critical value of positive samples according to the judgment standard X+2SD. Calculate the critical value again according to statistical principles. Its measurement results are shown in Table 2.

[0084] Table 2

[0085]

[0086]

[0087] As shown in Table 2, the average PI value was 19.94% with a standard deviation of 10.17%. Based on this, the PI cutoff value for the icELISA was calculated to be 40.28%. Samples with a PI value below 40.28% were considered negative, and those with values ​​below 40.28% were considered positive.

[0088] Validation case, sensitivity, specificity and repeatability test of indirect competitive ELISA method

[0089] The GAstV-2 positive serum (i.e., gosling serum infected with GAstV-2) was diluted according to the dilution ratio of 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256 and 1:512, and the GAstV-2 positive serum of each dilution was used as a serum sample. The indirect competitive ELISA method optimized in Example 1 was used to test the sensitivity of the method. The results were as follows: Figure 5A As shown. Figure 5A It can be seen that the upper limit of detection (LOD) of icELISA is 1:128, at which point the PI value is slightly higher than the critical value.

[0090] DTMUV (duck swan virus) positive serum, GPV (goose paramyxovirus) positive serum, ARV (avian reovirus) positive serum, H9N2-AIV (H9N2 subtype avian influenza virus) positive serum, DHAV-1 positive serum, DHAV-3 (duck hepatitis A virus type 1 and type 3) positive serum, NDV (Newcastle disease virus) positive serum, GAstV-1 (goose astrovirus type 1) positive serum and GAstV-2 positive serum were used as serum samples and tested according to the indirect competitive ELISA method optimized in Example 1 to test the specificity of the method. The results are as follows: Figure 5B As shown. Figure 5BAs shown, icELISA showed no cross-reaction to these sera, and all PI values ​​were below the critical value, indicating that the icELISA method based on the present invention is specific for detecting GAstV-2 in serum.

[0091] Six serum samples (three positive and three negative, numbered 1#, 2#, and 3# for the three positive serum samples and 4#, 5#, and 6# for the three negative serum samples) were selected for in vivo and inter-individual reproducibility testing, with each serum sample tested in triplicate. The results of the in vivo reproducibility test are shown in Table 3, and the results of the inter-individual reproducibility test are shown in Table 4. In Tables 3 and 4, the horizontal labels 1, 2, and 3 represent the PI values ​​obtained from three parallel tests of the corresponding serum samples, Mean represents the mean of the three PI values ​​of the serum samples with the same number, SD represents the standard deviation, and CV% represents the coefficient of variation. As can be seen from Tables 2 and 3, the in vivo reproducibility coefficient of variation (CV) ranged from 1.66% to 6.28%, and the inter-individual reproducibility CV ranged from 2.61% to 12.18%, indicating that the icELISA has good reproducibility.

[0092] Overall, these data indicate that the icELISA established in the present invention has good sensitivity, reliability, and reproducibility.

[0093] Table 3

[0094]

[0095] Table 4

[0096]

[0097] Application Examples

[0098] Sixty goose serum samples were collected and tested using the icELISA method constructed in the present invention (i.e., the optimized indirect competitive ELISA method described in Example 1) and conventional IFA (i.e., the IFA detection method described above). Ten-fold diluted goose serum was used as the primary antibody, and FITC-labeled goat anti-duck IgG (KPL) was used as the secondary antibody.

[0099] The test results are shown in Table 5. icELISA detected 47 samples as positive and 13 as negative, while IFA detected 39 samples as positive and 21 as negative. Compared with IFA, the icELISA had a positive concordance rate of 80.85% (38 of 47) and a negative concordance rate of 92.31% (12 of 13). Therefore, the overall concordance rate between icELISA and IFA was 83.33% (50 of 60).

[0100] Table 5

[0101]

[0102] The present invention uses the recombinant protein of goose astrovirus type 2 ORF2 transformed into host cells and induced to express as the immunogen to immunize mice, and obtains two monoclonal antibodies (A10D12 and A12G1) through screening, and identifies two new epitopes (1MADRA5 and 36 YKPQKLPMKA 45 Both epitopes are located in the relatively conserved N-terminal S domain of the ORF2 protein. Sequence alignment results also demonstrated that these two epitopes are highly conserved among goose astrovirus type 2 strains, while differing significantly from other astroviruses, including type 1 astrovirus. Western blotting, IFA, and immunohistochemistry results demonstrated that both mAbs specifically reacted with goose astrovirus type 2. The excellent reactivity and conservation of the epitopes recognized by these two mAbs make them suitable for serological detection of goose astrovirus type 2.

[0103] The present invention further establishes an indirect competitive ELISA method for detecting anti-GAstV-2 antibodies in goose serum by competing with an anti-GAstV-2 ORF2 protein mouse monoclonal antibody that interacts with the coated antigen (although the indirect competitive ELISA method is established using only A10D12 monoclonal antibody as a specific example, it should be noted that the establishment of an indirect competitive ELIS method using A12G1 monoclonal antibody is not denied here). This method has high detection sensitivity, the maximum dilution of detectable antibodies is 1:128, and the correlation with the IFA test results is 83.33%. In addition, the method has good repeatability and strong specificity. It only reacts with GAstV-2 serum and has no cross-reaction with other detected pathogen sera. Moreover, compared with the indirect EILSA method commonly used in the prior art, this method solves the problems that the specificity of the indirect EILSA method is greatly affected by the captured antigen, and the indirect EILSA method is not conducive to promotion and application due to the difficulty in obtaining anti-goose secondary antibodies, providing a more novel and effective direction for serological monitoring of GAstV-2 infection.

[0104] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0105] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0106] In addition, the various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for constructing a hybridoma cell line based on goose astrovirus type 2, characterized in that: The construction method comprises: S100, amplifying the genetic material of goose astrovirus type 2 using the amplification primers shown in SEQ ID No: 1 and SEQ ID No: 2 to obtain an amplified product; S200, inserting the amplified product obtained in step S100 into an expression vector to construct a recombinant plasmid; S300, transforming the recombinant plasmid obtained in step S200 into a host cell and inducing expression to obtain ORF2 recombinant protein; S400, using the ORF2 recombinant protein obtained in step S300 to immunize animals, taking spleen cells from the immunized animals and fusing them with myeloma cells, followed by subcloning and screening to obtain a hybridoma cell line based on goose astrovirus type 2.

2. The construction method according to claim 1, characterized in that The expression vector in step S200 is a prokaryotic expression vector pET-30a; and / or, The host cell in step S300 is selected from Escherichia coli BL21 cells; and / or, The animals in step S400 are selected from BALB / c mice.

3. A hybridoma cell line obtained by the construction method according to claim 1 or 2, characterized in that: The hybridoma cell line is hybridoma cell line A10D12, whose preservation number is CCTCCNO: C202506, and was deposited in the China Center for Type Culture Collection on March 26, 2025.

4. A hybridoma cell line obtained by the construction method according to claim 1 or 2, characterized in that: The hybridoma cell line is hybridoma cell line A12G1, whose preservation number is CCTCCNO: C202507, and was deposited in the China Center for Type Culture Collection on March 26, 2025.

5. A monoclonal antibody against goose astrovirus type 2 ORF2 protein, characterized in that: The monoclonal antibody against the ORF2 protein of type 2 goose astrovirus is prepared from the hybridoma cell line according to claim 3 or 4.

6. The monoclonal antibody against goose astrovirus type 2 ORF2 protein according to claim 5, characterized in that The heavy chain of the monoclonal antibody against type 2 goose astrovirus ORF2 protein is of IgG1 type, and the light chain is of κ type.

7. The monoclonal antibody against goose astrovirus type 2 ORF2 protein according to claim 5 or 6, characterized in that: The monoclonal antibody against type 2 goose astrovirus ORF2 protein prepared by hybridoma cell line A10D12 specifically recognizes the antigenic epitope of type 2 goose astrovirus ORF2 protein. 36 YKPQKLPMKA 45 .

8. The monoclonal antibody against goose astrovirus type 2 ORF2 protein according to claim 5 or 6, characterized in that: The antigenic epitope of the type 2 goose astrovirus ORF2 protein specifically recognized by the monoclonal antibody against the type 2 goose astrovirus ORF2 protein prepared by the hybridoma cell line A12G1 is 1MADRA5.

9. A detection reagent for goose astrovirus type 2, characterized in that: The detection reagent comprises the hybridoma cell line according to claim 3 or 4, or comprises the monoclonal antibody against the ORF2 protein of type 2 goose astrovirus according to any one of claims 5-8.

10. The detection reagent according to claim 9, characterized in that The detection reagent is an indirect competitive ELISA detection reagent; Preferably, the detection reagent uses the monoclonal antibody against the type 2 goose astrovirus ORF2 protein as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG as the secondary antibody.

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