Goose astrovirus type 1-based hybridoma cell strain, construction method, monoclonal antibody and detection reagent

Abstract

The invention discloses a hybridoma cell strain based on type 1 goose astrovirus, a construction method, a monoclonal antibody and a detection reagent. The hybridoma cell strain is a hybridoma cell strain A5A1, the preservation number of the hybridoma cell strain is CCTCC (China Center for Type Culture Collection) NO: C202505, and the hybridoma cell strain is preserved in the China Center for Type Culture Collection on March 26, 2025. According to the invention, a mouse is immunized by GAstV-1ORF2 recombinant protein expressed by a prokaryotic expression system, and a monoclonal antibody A5A1 is obtained by screening and subcloning. Indirect competitive ELISA (icELISA) for detecting the GAstV-1 antibody is developed based on the hybridoma cell strain A5A1, and the method is high in sensitivity, good in repeatability and high in specificity, only reacts with serum of GAstV-1, and does not have cross reaction with other detected pathogenic serum. A serological detection method established by the monoclonal antibody prepared by the method lays a foundation for scientific prevention and control of gout of goslings.

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 1, a construction method, a monoclonal antibody and a detection reagent. Background Art

[0002] Goose Astrovirus (GAstV) is a small, non-enveloped virus belonging to the genus Avian Astrovirus in the family Astroviridae. Based on gene homology and genetic distance analysis, goose astrovirus is divided into two gene groups, GAstV-1 and GAstV-2. The nucleotide homology of the GAstV genomes of these two branches is only about 60%, and the nucleotide homology of ORF2 is even lower. The GAstV-1 genome is a single-stranded positive-sense RNA molecule of approximately 7.2 kb in length, containing three open reading frames, ORF1a, ORF1b, and ORF2. ORF1a and ORF1b encode non-structural proteins, including a transmembrane domain (TM), a serine protease, and an RNA-dependent RNA polymerase. ORF2 encodes structural proteins that play a key role in the maturation and packaging of viral particles and the immune response. The astrovirus ORF2 structural protein forms four domains after intracellular and extracellular hydrolysis. Among them, the S and P1 domains located at the N-terminus are relatively conserved and constitute the inner layer of the virus particle. The P2 domain and the acidic terminal domain located at the C-terminus are highly variable and constitute the spike of the virus particle.

[0003] Studies have shown that the pathogens of gosling gout are GAstV-1 and GAstV-2. Although epidemiological surveys show that GAstV-2 is the main prevalent strain, GAstV-1 infection has been on the rise in recent years, and co-infection with GAstV-1 and GAstV-2 is becoming increasingly common. Vaccines for goose astrovirus infection are still under development. The prevention and control of gosling gout mainly relies on biosafety measures. Therefore, the establishment of efficient detection methods is particularly important. There are currently many reports on detection methods for goose astrovirus type 2 infection, such as real-time fluorescence quantitative PCR and loop-mediated isothermal amplification (LAMP) for antigen detection, and indirect ELISA and competitive ELISA for antibody detection. There are relatively few studies on detection methods for goose astrovirus type 1, especially monoclonal antibodies and serological methods.

[0004] Therefore, providing a monoclonal antibody for detecting goose astrovirus type 1 and a serological detection method constructed based on the monoclonal antibody is an urgent problem to be solved in the present invention. 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 1, with high sensitivity, high specificity and good reproducibility.

[0006] To achieve the above objectives, the present invention provides a method for constructing a hybridoma cell line based on goose astrovirus type 1, the method comprising:

[0007] S100, amplifying the genetic material of goose astrovirus type 1 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 a host cell 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, and then subcloning and screening to obtain a hybridoma cell line based on goose astrovirus type 1.

[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 above-described construction method, wherein the hybridoma cell line is hybridoma cell line A5A1, whose preservation number is CCTCC NO: C202505, and which was deposited in the China Center for Type Culture Collection on March 26, 2025.

[0015] The present invention also provides a monoclonal antibody against the ORF2 protein of goose astrovirus type 1, 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 1.

[0016] Preferably, the heavy chain of the monoclonal antibody against type 1 goose astrovirus ORF2 protein is of IgG1 type, and the light chain is of κ type.

[0017] Preferably, the antigenic epitope of the type 1 goose astrovirus ORF2 protein specifically recognized by the type 1 goose astrovirus ORF2 protein monoclonal antibody is 394 SNREVQITQL 403 .

[0018] The present invention also provides a detection reagent for goose astrovirus type 1, wherein the detection reagent comprises the hybridoma cell line described above, or comprises the monoclonal antibody against goose astrovirus type 1 ORF2 protein described above.

[0019] Preferably, the detection reagent is an indirect competitive ELISA detection reagent.

[0020] Preferably, the detection reagent uses the monoclonal antibody against the type 1 goose astrovirus ORF2 protein as the primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG as the secondary antibody.

[0021] The present invention immunized mice with the GAstV-1ORF2 recombinant protein expressed in a prokaryotic expression system, and obtained a monoclonal antibody (mAb) A5A1 through screening and subcloning. Western blot, IFA and immunohistochemistry results all showed that the monoclonal antibody A5A1 could specifically react with GAstV-1. At the same time, the present invention developed an indirect competitive ELISA (icELISA) for detecting GAstV-1 antibodies based on the hybridoma cell line A5A1. This method has high sensitivity, good repeatability, and strong specificity. It only reacts with the serum of GAstV-1 and has no cross-reaction with other pathogenic sera tested. The serological detection method established based on the monoclonal antibody prepared by the method of the present invention also laid the foundation for the scientific prevention and control of gout in goslings. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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:

[0023] Figure 1A This is a diagram showing the results of SDS-PAGE analysis of the ORF2 recombinant protein in Detection Example 1;

[0024] Figure 1B This is the result of Western blotting detection of purified ORF2 recombinant protein using anti-His tag antibody in Detection Example 1;

[0025] Figure 2A This is the result of IFA identification of GEK cells using monoclonal antibody A5A1 in detection example 2;

[0026] Figure 2BThis is a graph showing the results of Western blotting of GEK cells using monoclonal antibody A5A1 in Detection Example 2;

[0027] Figure 2C This is the result of immunohistochemical detection of GEK cells using monoclonal antibody A5A1 in detection example 2;

[0028] Figure 3A is a schematic diagram of the ORF2 protein domain of goose astrovirus type 1 in detection example 3;

[0029] Figure 3B This is the IFA test result of 293 cells transfected with different recombinant plasmids in Test Example 3;

[0030] Figure 4A This is an animated diagram of the B cell epitope analysis in ORF2 of the GAstV-1TZ03 structural protein recognized by the monoclonal antibody A5A1 in Detection Example 3;

[0031] Figure 4B This is a spheroid model diagram for the B cell epitope analysis in ORF2 of the GAstV-1TZ03 structural protein recognized by the monoclonal antibody A5A1 in Detection Example 3;

[0032] Figure 4C is a graph showing the comparison of the amino acid sequences of GAstV-1TZ03 and other astrovirus structural proteins in detection example 3;

[0033] Figure 5A This is the test result diagram of the sensitivity test in the verification case;

[0034] Figure 5B This is a graph showing the detection results of the specificity test in the verification example. DETAILED DESCRIPTION

[0035] 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.

[0036] The technical solutions of the present invention are described in detail below through specific examples. The GAstV-1TZ03 strain type 1 goose astrovirus 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.

[0037] The His tag antibody was a commercially available product from Kangwei Company in Taizhou, China; the RNA extraction kit was a commercially available MagicPure Up 32 Viral DNA / RNA Kit from Transgene Company in Beijing; the One-step RT-PCR Kit was a commercially available Titanium One-Step RT-PCR Kit (TaKaRa) from Takara Biotechnology (Dalian) Co., Ltd.; Sal I restriction endonuclease, Xho I restriction endonuclease, and T4 DNA ligase were commercially available products (TaKaRa) from Takara Biotechnology (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 determination 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., USA. 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.

[0038] Among them, in the present invention, the operation method of Western blotting identification is as follows:

[0039] After separation by 10% SDS-PAGE, the samples were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk powder for 1 hour at room temperature. Subsequently, reactions were performed using appropriately diluted His-tag antibodies or monoclonal antibodies as primary antibodies and horseradish peroxidase-conjugated goat anti-mouse IgG (KPL) antibodies as secondary antibodies. Specific bands were detected using an ECL kit (Vazyme).

[0040] Regarding IFA testing, the specific operation method is as follows:

[0041] 24-well cell plates of GEK cells infected with GAstV-1TZ03 strain or 293 cells transfected with GAstV-1ORF2 and its truncated fragments were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.05% Triton X-100 for 10 minutes, and then blocked with 3% BSA. Subsequently, the cell plates were incubated with diluted monoclonal antibodies at 37°C for 1 hour, washed with PBS, and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG (KPL) was added as a secondary antibody and incubated at 37°C for 1 hour. Finally, fluorescent cells were observed using an inverted fluorescence microscope.

[0042] Regarding IHC (immunohistochemistry), the specific operation methods are as follows:

[0043] One-day-old healthy goose chicks were orally inoculated with 0.3 mL of GAstV-1TZ03 strain (0.3 × 10 3.75 TCID 50 Each goose received an oral dose of the same volume of PBS. Five days after inoculation, the geese were euthanized with an intravenous injection of sodium pentobarbital. The small intestine, lungs, and heart were collected and fixed with 4% paraformaldehyde. Immunohistochemical analysis was performed using monoclonal antibodies (mAbs) as primary antibodies and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (KPL) as a secondary antibody.

[0044] 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 1 prepared in Preparation Example 3 of the present invention.

[0045] Preparation Example 1: Expression and purification of goose astrovirus type 1 ORF2 recombinant protein

[0046] Viral RNA of goose astrovirus type 1 GAstV-1TZ03 strain was extracted using an RNA extraction kit. One-step RT-PCR Kit and SEQ ID No: 1 (ORF2-F: 5'-TAAGAAGGAGATATACATATGGCCGACAAGGTCACTGTC-3') and SEQ ID No: 2 (ORF2-R:

[0047] The extracted RNA was amplified using the primers shown in Figure 5'-GCTTCCTTTCGGGCTTTGTTAATCAAACTCTTGTCCGCC-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.

[0048] The resulting recombinant plasmid pET-ORF2 was transformed into competent 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 100 ng / μL.

[0049] Preparation Example 2: Construction of a hybridoma cell line for preparing monoclonal antibodies against goose astrovirus type 1 ORF2 protein

[0050] 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.

[0051] The spleen cells of 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 to obtain a positive hybridoma cell line. The obtained positive hybridoma cell line was further subjected to three rounds of subcloning to ensure its monoclonality, and a hybridoma cell line stably expressing a monoclonal antibody against the GAstV-1 ORF2 protein was successfully obtained and named hybridoma cell line A5A1.

[0052] Preparation Example 3: Preparation of Monoclonal Antibodies Against Type 1 Goose Astrovirus ORF2 Protein

[0053] 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 A5A1 (≥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 a highly pure (>90%) monoclonal antibody, designated as mAb A5A1.

[0054] Detection Example 1: Identification of ORF2 recombinant protein

[0055] 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 and Figure 1B shown. Specifically:

[0056] 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.

[0057] Figure 1B The figure shows the results of Western blotting detection of the purified ORF2 recombinant protein using an anti-His tag antibody (i.e., His-tag monoclonal antibody); wherein, band M is a molecular weight marker, band 1 is the uninduced bacterial liquid after transformation with pET-ORF2, and band 2 is the purified ORF2 recombinant protein in Preparation Example 1.

[0058] 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. The ORF2 recombinant protein was successfully purified using anti-His magnetic beads. Figure 1B As shown, the purified ORF2 recombinant protein obtained in Preparation Example 1 can be recognized and bound by the anti-His tag monoclonal antibody, verifying that the ORF2 recombinant protein obtained in Preparation Example 1 was successfully expressed in Escherichia coli.

[0059] Test Example 2: Identification of Monoclonal Antibodies Targeting ORF2 Protein of Goose Astrovirus Type 1

[0060] The monoclonal antibody A5A1 prepared in Preparation Example 3 was identified using a typing ELISA kit. The identification showed that the heavy chain of the hybridoma cell line A5A1 was IgG1 type and the light chain was κ type.

[0061] The monoclonal antibody A5A1 prepared in Preparation Example 3 was used to detect GEK cells infected with GAstV-1 by IFA, Western blotting and IHC, and the results were as follows: Figure 2A-2C As shown. Among them, Figure 2A The results of IFA identification of GEK cells using monoclonal antibody A5A1 are shown in Figure 2. Figure 2A Middle, the left panel shows GEK cells not infected with GAstV-1 as a negative control, and the right panel shows GEK cells infected with GAstV-1 detected by monoclonal antibody A5A1; Figure 2B The figure shows the results of Western blotting detection of GEK cells using monoclonal antibody A5A1. Figure 2B In the middle, lane 1 is the detection of GAstV-1-infected GEK cells by monoclonal antibody A5A1, and lane 2 is the GEK cells not infected with GAstV-1 as a negative control; Figure 2C The results of IHC test were shown. The intestine, lung and heart tissues of goose chicks infected and not infected with GAstV-1TZ03 were reacted with monoclonal antibody A5A1 ( Figure 2C In the middle, the upper row shows uninfected goslings, and the lower row shows goslings infected with GAstV-1TZ03).

[0062] Figure 2A The IFA results showed that the monoclonal antibody A5A1 prepared by the present invention can specifically recognize GEK cells infected with GAstV-1; Figure 2B Western blotting results showed that the monoclonal antibody A5A1 prepared in the present invention could react specifically with GEK cell lysates infected with GAstV-1, and the band size was about 70 kD; Figure 2C The IHC test results showed that monoclonal antibody A5A1 could specifically bind to viral particles in the small intestine, lung and heart tissues of goslings infected with GAstV-1, while no positive signals were seen in the control group.

[0063] The above results indicate that the monoclonal antibody A5A1 prepared in the present invention can specifically recognize and bind to GAstV-1 ORF2 structural protein and virus particles, and can be used for the detection of GAstV-1 antigens.

[0064] Detection Example 3: B cell epitope localization and analysis

[0065] According to the three domains of the GAstV-1ORF2 gene, namely S, P1 and P2, three fragments of the GAstV-1TZ03 ORF2 gene were amplified, and then the N-terminus of the P1 gene fragment was truncated to varying degrees to obtain P1 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 eukaryotic expression vector pSC-Flag using a cloning 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. 48 hours after transfection, the monoclonal antibody A5A1 prepared in Preparation Example 3 was used as the primary antibody and FITC-labeled goat anti-mouse antibody was used as the secondary antibody, and an indirect immunofluorescence assay (IFA) was performed according to conventional methods.

[0066] Table 1

[0067]

[0068] Schematic diagram of the ORF2 protein domain of goose astrovirus type 1 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 logo at the top refers to the marker of the ORF2 gene fragment contained in the recombinant plasmid used to transfect 293 cells, and the logo on the left, from top to bottom, respectively, represents the positive control and monoclonal antibody A5A1 as the primary antibody.

[0069] pass Figure 3A and Figure 3B It can be seen that mAb A5A1 reacts specifically with 293 cells transfected with P1 gene fragment, but does not react with 293 cells transfected with S or P2 gene fragment, indicating that the epitope recognized by mAb A5A1 is located in the P1 domain of ORF2 protein. Further results showed that mAb A5A1 can recognize P1-140 gene fragment but not P1-130 gene fragment, indicating that the B cell epitope recognized by mAb A5A1 is located in the P1 domain. 131 SNREVQITQL 140 , corresponding to the ORF2 structural protein 394 SNREVQITQL 403 .

[0070] The 3D structure of the P1 domain of the GAstV-1 ORF2 structural protein was predicted using the online analysis tool Phyre, and the B cell epitopes recognized by the monoclonal antibody were visualized using the software PyMOL. Figure 4A and Figure 4B As shown (specifically, the three-dimensional structural model diagram of GAstV-1TZ03 ORF2 structural protein generated by PyMOL, Figure 4A For animated pictures, Figure 4B (Figure 2 is a spherical model diagram), as shown in the figure, the B cell epitope recognized by A5A1 131 SNREVQITQL 140 Located on the surface of the P1 domain.

[0071] The amino acid sequences of astrovirus ORF2 structural proteins were downloaded from the GenBank database, and the sequences of astrovirus ORF2 structural proteins were aligned using LasergeneMegAlign software to analyze B cell epitopes. 131 SNREVQITQL 140 Conservation between GAstV-1 and other astrovirus strains. Figure 4C The comparison results show that the B cell epitope of GAstV-1TZ03 recognized by A5A1131 SNREVQITQL 140 It is completely consistent with other GAstV-1 strains, but only shares 2-4 amino acids with other astrovirus strains, indicating that the B cell epitope recognized by monoclonal antibody A5A1 is highly conserved among GAstV-1 strains, but very different from other astrovirus strains, including GAstV-2 strains.

[0072] Example 1. Establishment and optimization of indirect competitive ELISA (icELISA) method

[0073] The purified ORF2 recombinant protein from Preparation Example 1 was diluted in 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 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. 50 μL of monoclonal antibody A5A1 diluted in blocking buffer (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, and 1:512 for a single variable multi-step gradient experiment) was added to each well and incubated at 37°C for 1-2 h. After washing five times with PBST, 100 μL of HRP-conjugated goat anti-mouse IgG secondary antibody was added 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). The wells were incubated at 37°C for 1 hour and washed five times with PBST. 100 μL of colorimetric solution was added to each well and incubated at 37°C in the dark for 5–30 minutes. The reaction was terminated by adding 50 μL of 2 mol / L sulfuric acid. OD values ​​were read at 450 nm using a Bio-Rad microplate reader. Optimal reaction parameters were optimized based on the maximum negative / positive (N / P) ratio.

[0074] The optimal reaction parameters finally determined after optimization were as follows: 100 μL of 1 μg / mL purified ORF2 recombinant protein in Preparation Example 1 was coated on a 96-well plate and incubated at 4°C for 16 hours; the plate was blocked with 5% skim milk powder at 37°C for 30 minutes; 100 μL of the serum sample to be tested diluted 1:16 was mixed with 100 μL of monoclonal antibody A5A1 diluted 1:2000 and then competitively reacted at 37°C for 2 hours; the plate was incubated with a 1:10000 dilution of horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody at 37°C for 1 hour; the TMB substrate was reacted at 37°C for 15 minutes; and 50 μL of 2 mol / L H2SO4 was added to terminate the reaction.

[0075] Example 2: Determination of critical value of indirect competitive ELISA method

[0076] Utilize the icELISA conditions optimized in Example 1 to detect 30 GAstV-1 clinical negative sera, measure its absorbance at 450nm, and calculate the inhibition rate (PI) according to the formula. Calculate the average inhibition rate (X) and standard deviation (SD) of 30 negative sera, 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.

[0077] Table 2

[0078]

[0079] As shown in Table 2, the average PI value of the 30 negative serum samples was 18.98%, with a standard deviation of 9.89%. According to the calculation formula, the PI cutoff value of the icELISA is 38.76% (Table 2). Therefore, serum samples with a PI value below 38.76% were considered negative, and those with a PI value below 38.76% were considered positive.

[0080] Validation case, sensitivity, specificity and repeatability test of indirect competitive ELISA method

[0081] The GAstV-1 positive serum (i.e., gosling serum infected with GAstV-1) 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-1 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 PI value of the positive serum diluted 1:128 is 52.81%, while the PI value of the positive serum diluted 1:256 is 34.75%, indicating that the lower limit of detection (LOD) of icELISA is 1:128.

[0082] DTMUV (duck Tembusu virus) positive serum, GPV (goose parvovirus) positive serum, ARV (avian reovirus) positive serum, H9N2-AIV (H9N2 subtype avian influenza virus) positive serum, DHAV-1 and DHAV-3 (duck hepatitis A virus type 1 and type 3) positive serum, NDV (Newcastle disease virus) positive serum and GAstV-2 (goose astrovirus type 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 5BIt can be seen that all PI values ​​were below the critical value, indicating that icELISA did not show cross-reactivity to these sera and the method had good specificity for GAstV-1.

[0083] 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 intra- and inter-batch reproducibility testing, with each serum sample tested in triplicate. The results of the intra-batch reproducibility test are shown in Table 3, and the results of the inter-batch 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 ​​for the serum samples with the same number, SD represents the standard deviation, and CV% represents the coefficient of variation. As shown in Tables 3 and 4, the coefficient of variation (CV) for intra-batch reproducibility ranged from 1.74% to 4.53%, and the CV for inter-batch reproducibility ranged from 2.80% to 9.95%, indicating that the icELISA has good reproducibility.

[0084] Overall, the above data show that the icELISA of the present invention has good sensitivity, specificity and reproducibility.

[0085] Table 3

[0086] Sample number 1 2 3 Mean SD CV% 1# 90.95％ 92.53％ 95.69％ 93.05％ 0.0241 2.59 2# 87.21％ 86.54％ 81.26％ 85.00％ 0.0326 3.84 3# 74.60％ 72.25％ 75.50％ 74.11％ 0.0168 2.26 4# 3.22％ 3.10％ 3.14％ 3.15％ 0.0006 1.81 5# 12.19％ 11.17％ 11.92％ 11.76％ 0.0053 4.53 6# 5.03％ 4.96％ 5.13％ 5.04％ 0.0009 1.74

[0087] Table 4

[0088] Sample number 1 2 3 X SD CV% 1# 91.98％ 85.59％ 89.83％ 89.13％ 0.03253 3.65 2# 89.24％ 80.52％ 84.59％ 84.78％ 0.04364 5.15 3# 72.50％ 76.55％ 73.69％ 74.25％ 0.02081 2.80 4# 12.60％ 10.58％ 11.01％ 11.39％ 0.01063 9.33 5# 6.02％ 6.57％ 5.61％ 6.07％ 0.0048 7.92 6# 4.32％ 3.92％ 3.54％ 3.92％ 0.0039 9.95

[0089] Application Examples

[0090] Eighty 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.

[0091] The test results are shown in Table 5. The icELISA detected 35 samples as positive and 45 as negative, while the IFA detected 29 samples as positive and 51 samples as negative. Compared with the IFA, the icELISA had a positive concordance rate of 77.14% (27 / 35) and a negative concordance rate of 95.56% (43 / 45). Therefore, the overall concordance rate between the icELISA and IFA was 87.5% (70 / 80).

[0092] Table 5

[0093]

[0094] The present invention immunized mice with the recombinant protein of goose astrovirus type 1 ORF2 expressed in Escherichia coli, and obtained a hybridoma cell line A5A1 after screening. Immunofluorescence experiments (IFA) and immunohistochemistry experiments (IHC) confirmed its specificity for GAstV-1 infected tissues, and Western blotting analysis detected two specific protein bands in infected cells, which were approximately 90kDa and 70kDa, respectively. These molecular weights correspond to the immature and mature capsid proteins observed in human astroviruses (HAstVs). Specifically, the mature protein VP70 (about 70kDa) is produced by proteolytic cleavage of the immature precursor VP90 (about 90kDa) by host proteases. Therefore, it further highlights the application value of the hybridoma cell line A5A1 obtained by the present invention in detecting virus particles and their processed forms.

[0095] At the same time, the present invention is based on the obtained monoclonal antibody against goose astrovirus type 1, and establishes an indirect competitive ELISA method for detecting anti-GAstV-1 antibodies in goose serum by competing with monoclonal antibody A5A1 for binding to the coating antigen. This method has high repeatability and strong specificity, and has extremely low cross-reactivity with other common waterfowl virus antisera. The maximum dilution factor of the antibody that can be detected by this method is 1:128, which is lower than the sensitivity of the indirect ELISA (1:6400) established by Zhang et al. using goose astrovirus type 1 as the coating antigen. The test results showed that the detection rate of goose astrovirus type 1 antibodies was 43.75% (35 / 80), which is comparable to the detection rate of the indirect ELISA method (33.3%), indicating that the indirect competitive ELISA method established by the present invention is a promising serological diagnostic tool that can be used for serological monitoring of GAstV-1 infection in geese.

[0096] 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.

[0097] 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.

[0098] 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 1, characterized in that: The construction method comprises: S100, amplifying the genetic material of goose astrovirus type 1 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 host cells 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, and then subcloning and screening to obtain a hybridoma cell line based on goose astrovirus type 1.

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 A5A1, whose preservation number is CCTCCNO: C202505, and was deposited in the China Center for Type Culture Collection on March 26, 2025.

4. A monoclonal antibody against goose astrovirus type 1 ORF2 protein, characterized in that: The monoclonal antibody against goose astrovirus type 1 ORF2 protein is prepared from the hybridoma cell line according to claim 3.

5. The monoclonal antibody against goose astrovirus type 1 ORF2 protein according to claim 4, characterized in that The heavy chain of the monoclonal antibody against type 1 goose astrovirus ORF2 protein is of IgG1 type, and the light chain is of κ type.

6. The monoclonal antibody against goose astrovirus ORF2 protein type 1 according to claim 4 or 5, characterized in that The antigenic epitope of the type 1 goose astrovirus ORF2 protein specifically recognized by the type 1 goose astrovirus ORF2 protein monoclonal antibody is 394 SNREVQITQL 403 .

7. A detection reagent for goose astrovirus type 1, characterized in that: The detection reagent comprises the hybridoma cell line according to claim 3, or comprises the monoclonal antibody against the ORF2 protein of type 1 goose astrovirus according to any one of claims 4 to 6.

8. The detection reagent according to claim 7, characterized in that The detection reagent is an indirect competitive ELISA detection reagent.

9. The detection reagent according to claim 8, characterized in that The detection reagent uses the monoclonal antibody against the ORF2 protein of type 1 goose astrovirus as a primary antibody and horseradish peroxidase-labeled goat anti-mouse IgG as a secondary antibody.

Images