A goose type 2 astrovirus monoclonal antibody and an antigen epitope peptide thereof
By preparing monoclonal antibodies against type 2 goose astrovirus and their antigenic epitope peptides, the problem of efficient serological detection has been solved, enabling specific recognition and immune response monitoring of type 2 goose astrovirus, and supporting virus research and vaccine development.
Patent Information
- Application Number
- CN202510027590.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-08
- Publication Date
- 2026-02-10
- Estimated Expiration
- 2045-01-08
AI Technical Summary
Currently, there is a lack of efficient serological detection methods for type 2 goose astrovirus, the pathogenic mechanism is unclear, and it is impossible to effectively monitor and prevent the spread of the virus.
Monoclonal antibodies against type 2 goose astrovirus and their antigenic epitope peptides were prepared. The monoclonal antibodies were obtained by immunizing mice, and their corresponding antigenic epitopes were identified. These antibodies were then applied to the preparation of serological diagnostic tools and epitope vaccines.
It provides an efficient serological diagnostic method that can specifically identify type 2 goose astrovirus, support research on viral structure and function, and enhance the ability to monitor immune responses to the virus.
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Figure CN119735673B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular biology technology, specifically to a type 2 goose astrovirus monoclonal antibody and its antigenic epitope peptide. Background Technology
[0002] Currently, the pathogenic mechanism and immune response of goose astrovirus (GAstV) are still unclear, and there is a lack of efficient serological detection methods for GAstV.
[0003] Goose astrovirus belongs to the family Astroviridae and the genus *Gastrovirus*. Its genome consists of two non-coding regions (5'UTR and 3'UTR), three open reading frames (ORF1a, ORF1b, ORF2), and a poly A tail, with a genome length of approximately 7200 nt. ORF1a and ORF1b encode its non-structural proteins (nsp1a and nsp1b), influencing GAstV replication and transcription. GAstV ORF2 encodes the viral structural protein (Cap protein), an antigenic determinant of GAstV, capable of stimulating an immune response and serving as the primary antigen inducing protective antibodies. It is closely related to GAstV cell receptor recognition, host innate immune responses, and cell proliferation. Goose astrovirus particles have a typical star-shaped morphology, formed by protrusion proteins on the viral surface. The GAstV viral capsid is composed of a single type of structural protein, which forms a robust outer shell on the viral particle, protecting the viral genetic material. This capsid is called the "Cap protein." GAstV virus particles typically have a diameter between 28 and 30 nanometers. According to the ICTV classification, different genotypes of goose astroviruses are mainly classified into goose astrovirus type 1 and type 2 based on their ORF2 (Cap) genotype. Due to differences in genomic structure, different genotypes of goose astroviruses exhibit varying degrees of pathogenicity and host adaptability. Summary of the Invention
[0004] The purpose of this invention is to solve the above-mentioned technical problems and provide a type 2 goose astrovirus monoclonal antibody and its antigenic epitope peptide.
[0005] The technical solution adopted in this invention is as follows:
[0006] One of the objectives of this invention is to provide a monoclonal antibody against type 2 goose astrovirus, wherein the monoclonal antibody is prepared by immunizing mice with the capsid protein GAstVCap of type 2 goose astrovirus as an antigen; the monoclonal antibody includes a heavy chain variable region and a light chain variable region, and the amino acid sequences are shown in SEQ ID No. 3 and SEQ ID No. 5, respectively.
[0007] Preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 type, and the light chain constant region is of the Kappa type.
[0008] Preferably, the monoclonal antibody further includes a heavy chain constant region and a light chain constant region, with amino acid sequences as shown in SEQ ID No. 7 and SEQ ID No. 9, respectively.
[0009] A second objective of this invention is to provide a recombinant expression plasmid containing a gene fragment encoding the type 2 goose astrovirus capsid protein GAstV Cap, the nucleotide sequence of which is shown in SEQ ID No. 1.
[0010] A third objective of this invention is to provide a host cell comprising the recombinant expression vector as described in claim 3.
[0011] The fourth objective of this invention is to provide an antigenic epitope peptide that specifically binds to the type 2 goose astrovirus monoclonal antibody described in claim 1; the amino acid sequence of the antigenic epitope peptide is shown in SEQ ID No. 10.
[0012] The fifth objective of this invention is to provide the application of the above-mentioned type 2 goose astrovirus monoclonal antibody in the preparation of goose astrovirus serological diagnostic reagents.
[0013] The sixth objective of this invention is to provide the application of the above-mentioned antigenic epitope peptide in the preparation of type 2 goose astrovirus epitope vaccine.
[0014] The beneficial effects of this invention are as follows: This invention prepares a monoclonal antibody against GAstV type 2, named 1F1, by immunizing mice with GAstV capsid protein as an antigen; simultaneously, its corresponding antigenic epitope is identified, and this antigenic epitope is relatively conserved. Therefore, the monoclonal antibody and its antigenic epitope in this invention can be applied to the study of the structure and function of GAstV capsid protein, serological diagnostic tools, and the preparation of epitope vaccines. Attached Figure Description
[0015] Figure 1 The results of enzyme digestion electrophoresis of the GAstV-Cap recombinant expression plasmid constructed in this invention and the results of SDS-PAGE after purification of the recombinant protein are shown.
[0016] Figure 2 Western blot verification of GAstV Cap prokaryotic expression.
[0017] Figure 3 The image shows the antibody titer of mice immunized with recombinant GAstV Cap protein by ELISA. The left, right, middle, front, back, and none represent mice immunized 1, 2, 3, 4, 5, and 6.
[0018] Figure 4 For the detection of monoclonal antibody recognition ability, (A) the IFA method was used to detect monoclonal antibody recognition ability; (B) the indirect ELISA method was used to detect monoclonal antibody recognition ability; and (C) the WB method was used to detect monoclonal antibody recognition ability.
[0019] Figure 5 A schematic diagram showing the results of the monoclonal antibody 1F1 recognizing a truncated fragment of the GAstV Cap protein.
[0020] Figure 6 Western blot diagrams showing the precise localization of the 1F1 epitope of the monoclonal antibody, where (A) uses a GST-tagged antibody to verify the expression of the truncated protein; and (B) uses the 1F1 monoclonal antibody to locate the epitope.
[0021] Figure 7 This is a diagram showing the conservation of the GAstV Cap protein epitope sequence through multiple sequence alignment.
[0022] The type 2 goose astrovirus in this invention was preserved and provided by the Poultry Disease Center Laboratory of Sichuan Agricultural University, with the viral genome accession number GenBank: OQ909424.1. Detailed Implementation
[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0024] Example 1
[0025] 1. Construction, induction of expression, and purification of GAstV Cap protein prokaryotic expression plasmid
[0026] 1.1 Construction of full-length prokaryotic expression plasmid for GAstV Cap protein
[0027] The pET-28a(+) plasmid was digested with two restriction endonucleases, XhoⅠ and BamHI, to obtain a linear vector. Using goose astrovirus type 2 as a template, viral RNA was extracted and reverse transcribed into cDNA. The full-length Cap protein gene fragment was amplified using primers SEQ No. 36 and SEQ No. 37. The amplified fragment was then ligated into the pET-28a(+) linear vector using homologous recombinase. A 6×His tag was added to the N-terminus of the recombinant fragment. The ligation product was transformed into DH5α competent cells. The correctly sequenced recombinant plasmid was named pET-28a-Cap-His. The plasmid was then transformed into BL21 competent cells, followed by colony PCR identification and sequencing. After confirming correct sequencing results, the bacterial strain was preserved for future use. PCR identification results are shown below. Figure 1 As shown in (A).
[0028] 1.2 Induced Expression and Identification of Recombinant Proteins
[0029] The preserved bacterial strain was inoculated into 5 mL of LB liquid medium containing 50 μg / mL ampicillin (Amp) at a final concentration of 50 μg / mL. The culture was incubated overnight at 37°C and 180 rpm. The next day, the strain was inoculated at a 1:50 ratio into 10 mL of LB liquid medium containing 50 μg / mL ampicillin (Amp) at a final concentration of 50 μg / mL. The culture was incubated at 37°C and 180 rpm until the OD value reached approximately 0.6. The culture was then divided into two groups, with 5 mL in each group. The first group received no treatment. The second group was treated with isopropyl β-D-1-Thio galactopyranoside (IPTG) at a final concentration of 1 mmol / L, and induced for expression at 37°C and 180 rpm for 6 h. After induction, the cells were centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in equilibration buffer. Then, 1% lysozyme (100 mg / mL) was added, and the cells were subjected to three freeze-thaw cycles before sonication. After disruption, the cells were centrifuged at 4°C and 12000 rpm for 10 min, and the precipitate was dissolved in equilibration buffer containing 8 M urea. The supernatants and precipitates from both groups were collected for analysis using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
[0030] 1.3 Recombinant protein induction and purification
[0031] (1) Massive induction of recombinant proteins
[0032] The preserved bacterial strain was inoculated into 10 mL of LB liquid medium with a final Amp concentration of 50 μg / mL and cultured overnight at 37°C and 180 rpm. The next day, it was inoculated into 1000 mL of LB liquid medium at a 1:50 ratio with a final Amp concentration of 50 μg / mL and cultured at 37°C and 180 rpm until the OD value was approximately 0.6. IPTG was then added to induce expression for 6 h. After induction, the culture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the culture was resuspended in equilibration buffer. 1% lysozyme (100 mg / mL) was added and the culture was subjected to three freeze-thaw cycles. The culture was then sonicated and centrifuged at 12000 rpm for 10 min at 4°C to collect the inclusion body precipitate.
[0033] (2) Purification of recombinant proteins
[0034] The collected inclusion body precipitate was resuspended and dissolved in a bacterial cell: lysate (containing 8M urea) ratio of 1:10, and purified according to the Ni NTABeads 6FF gravity column instruction manual. The purified sample was then subjected to SDS-PAGE electrophoresis. The purification effect was assessed based on the electrophoresis results, as shown below. Figure 1 As shown in (B).
[0035] (3) Refolding of inclusion body proteins
[0036] The dialysis bag containing inclusion bodies was placed in a refolding buffer for gradient refolding. The concentrations of urea in the refolding buffer were 6M, 4M, 2M, 1M and 0M, respectively. The dialysis time for each concentration was 6 hours. Dialysis was performed at 4°C. This gradual decrease in urea concentration achieved the effects of refolding and protecting proteins.
[0037] (4) Determination of purified protein concentration
[0038] The concentration of the purified protein was determined according to the Solarbio BCA Protein Concentration Assay Kit instructions.
[0039] Example 2
[0040] 2. Preparation and identification of GAstV Cap protein monoclonal antibodies
[0041] 2.1 Animal Immunization
[0042] Mice were immunized with the purified Cap recombinant protein obtained in Example 1. Six 6-week-old female BALB / c mice were used, with six immunized and one serving as a control. For immunization, 500 μL of 0.1 mg / mL Cap recombinant protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into six mice. A second immunization was performed 14 days after the initial immunization. A third immunization was performed 14 days later, following the same procedure as the initial immunization. Seven days after the third immunization, 50 μL of blood from the infraorbital sinus was collected and incubated at 37°C for 1 hour to allow for serum extraction. The serum was then centrifuged at 3000 rpm for 10 minutes at 4°C. The serum was serially diluted and its titer was detected by indirect ELISA. Three days before cell fusion, mice with the highest titers were selected for a booster immunization via intraperitoneal injection of 100 μg Cap protein.
[0043] 2.2 Indirect ELISA detection of mouse serum titers
[0044] The optimal antigen coating concentration was determined using an indirect ELISA array assay, and this concentration was used to coat Cap protein for detecting mouse serum titers. 1 mg / mL Cap recombinant protein was serially diluted from 1:100 to five gradients: 10 μg / mL, 5 μg / mL, 2.5 μg / mL, 1.25 μg / mL, and 0.625 μg / mL, with 100 μL per well, and incubated overnight at 4°C. After discarding the coating solution, 200 μL of PBS buffer (PBST) supplemented with 0.01% Tween-20 was added to each well for shaking and washing, followed by patting dry. This was repeated three times, for 10 min each time. Blocking was performed with 5% bovine serum albumin (BSA) diluted in PBST at 37°C for 2 h, followed by three more washes. Immunized mouse serum was serially diluted with PBS at dilutions of 1:1000, 1:2000, 1:4000, 1:8000, and 1:16000, with 100 μL per well. Serum from BALB / c mice was used as the negative control. The mixture was incubated at 37°C for 1 h. After discarding the primary antibody and washing as before, 100 μL of 1:3000 diluted HRP-goat anti-mouse IgG was added to each well. The mixture was incubated at 37°C for 1 h, followed by three repeated washes. After these steps, 100 μL of 3,3'-5,5'-tetraphenylamine (TMB) was added as the substrate for HRP, and the reaction was carried out at 37°C in the dark for 15 min. 50 μL of 2M H2SO4 stop solution was added to each well. The absorbance at 450 nm (OD450nm) was then measured using a microplate reader. The results are shown below. Figure 3 As shown.
[0045] 2.3 Preparation of myeloma cells (SP2 / 0) and feeder cells
[0046] Myeloma cells (SP2 / 0) were removed from liquid nitrogen, thawed in a 37°C water bath, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and transferred to T25 cell culture flasks. When the cell density reached approximately 90%, passage was performed. Since myeloma cells have weak adherence, trypsin digestion is not necessary. At least six T25 cell culture flasks containing healthy SP2 / 0 cells should be prepared before cell fusion.
[0047] Take one healthy 6-8 week old BALB / c mouse, euthanize it by cervical dislocation and immerse it in 75% alcohol for 10 minutes. Fix its limbs in a laminar flow hood, cut the skin to expose the peritoneum while ensuring the peritoneum remains intact, and wipe the peritoneum with an alcohol swab. Using a 10mL sterile syringe, carefully inject 10mL of pre-cooled HAT culture medium into the mouse's peritoneal cavity. Gently massage the abdomen with an alcohol swab to mix the culture medium in the peritoneal cavity, then aspirate the fluid from the peritoneal cavity and place it in a 50mL sterile centrifuge tube. After dilution, add the diluted solution to a 96-well cell culture plate.
[0048] 2.4 Preparation of spleen cells for immunization of mice
[0049] Mice that had received boosted immunization were euthanized by cervical dislocation and immersed in 75% alcohol for 10 minutes. They were then fixed with their abdomens facing upwards on a dissecting board in a laminar flow hood. Under sterile conditions, the skin and peritoneum of the mice's abdomen were cut open, the spleen was removed, ground with a grinding rod, resuspended in basal culture medium, filtered through a cell sieve, centrifuged at 1000 r / min at room temperature for 10 minutes, the supernatant was discarded, and the resuspended spleen was prepared in HAT culture medium for later use.
[0050] 2.5 Cell Fusion
[0051] The SP2 / 0 cells and spleen cells prepared above were mixed in a 1:5 ratio in a 50 mL centrifuge tube. After mixing, the mixture was centrifuged at 1500 rpm for 10 min at room temperature. The supernatant was discarded, and the cell pellet was loosened by gently shaking or tapping the bottom of the centrifuge tube. The centrifuge tube containing the mixed cells was placed in a 37°C water bath. 1 mL of 50% PEG1500 preheated to 37°C was slowly added dropwise over 1 min while gently shaking the tube to promote cell fusion. Then, 25 mL of preheated HAT medium was added dropwise over 1.5 min, starting slowly and then increasing the speed, to terminate the reaction. The centrifuge tube was then placed in a 37°C, 5% CO2 incubator and incubated for 5 min. After centrifugation at 1000 rpm for 10 min at room temperature, the supernatant was discarded, and the cells were resuspended in HAT complete medium. The cells were then aliquoted into 96-well plates and cultured. After 8 days, the medium was replaced with HT medium.
[0052] 2.6 Screening and subcloning of positive hybridoma cells
[0053] When the cell density reaches 1 / 4 of the bottom area of the well, the culture supernatant of hybridoma cells in each well is taken as the primary antibody for indirect ELISA detection. Hybridoma cells in wells with higher OD450nm values are subcloned and expanded for culture.
[0054] Subcloning was performed using a limiting dilution method. After three subcloning supernatants yielded positive hybridoma cells, an indirect ELISA method was used to screen for positive hybridoma cells, resulting in a hybridoma cell line that could secrete specific monoclonal antibodies and be stably passaged. This monoclonal antibody was named 1F1.
[0055] Example 3
[0056] 3. Identification of biological characteristics of monoclonal antibodies
[0057] 3.1 Indirect ELISA assay for detecting monoclonal antibody recognition ability
[0058] The PGEX-4T-1 plasmid was digested with XhoI and EcoRI restriction endonucleases to obtain a linear vector. Using pET-28a-Cap-His constructed in Example 1 as a template, the full-length Cap protein gene fragment was amplified using primers SEQ No. 11 and SEQ No. 12. Then, the amplified fragment was ligated into the PGEX-4T-1 linear vector using homologous recombinase. The ligation product was transformed into DH5α competent cells. The correctly sequenced recombinant plasmid was named PGEX-4T-1-Cap, and then transformed into BL21 competent cells. Colony PCR identification and sequencing were performed. After confirming the sequencing results, the bacterial strain was preserved. Expression of the strain was induced, and the expressed Cap protein was used as a coating antigen to coat ELISA plates. The GST protein of PGEX-4T-1 served as a negative control. The supernatant of the hybridoma cell culture was used as the primary antibody, and the monoclonal antibody recognition ability was detected by indirect ELISA. The specific method is described in section 2.2 of Example 2. The results showed that the monoclonal antibody recognition ability was significant. Figure 4 As shown in (B).
[0059] 3.2 Western blotting (WB) detection of monoclonal antibody recognition ability
[0060] Cap protein expressed in prokaryotic cells as PGEX-4T-1-Cap was subjected to electrophoresis on a 10% SDS-PAGE gel, followed by transfer to a polyvinylidene fluoride (PVDF) membrane using a semi-dry transfer system. The supernatant from hybridoma cell line 1F1 was used as the primary antibody, and horseradish peroxidase (HRP) conjugated with goat anti-mouse IgG was used as the secondary antibody. Protein detection was performed using ECL chemiluminescence. Results showed significant recognition ability of the monoclonal antibody. Figure 4 As shown in (C).
[0061] 3.3 Indirect immunofluorescence assay (IFA) to detect monoclonal antibody recognition ability
[0062] The PCAGGS plasmid was digested with EcoRI and BglII restriction endonucleases to obtain a linear vector. Using pET-28a-Cap-His constructed in Example 1 as a template, the full-length Cap protein gene fragment was amplified using primers SEQ No. 38 and SEQ No. 39. The amplified fragment was then ligated to the PCAGGS linear vector using homologous recombinase. The ligation product was transformed into DH5α competent cells. The correctly sequenced recombinant plasmid was named PCAGGS-Cap. BHK-21 cells were seeded in 12-well plates and transfected with PCAGGS-Cap for 48 h. Cells were washed with PBST and fixed with 4% paraformaldehyde at 4°C for 1 h or overnight. Cells were then infiltrated with 0.3% Triton X-100 in PBS at 4°C for 1 h. After blocking in 5% BSA at 37°C for 1 h, cells were treated with hybridoma cell culture supernatant at 37°C for 2 h. Cells were then incubated with FITC-labeled goat anti-mouse IgG (1:1000 dilution) at 37°C for 1 h, and stained with 4',6-diamidinyl-2-phenylindole dihydrochloride (DAPI) at room temperature for 10 min. After each step, the cells were washed three times with PBST for 10 min each time. Finally, the cells were observed and photographed under a fluorescence microscope. The results showed significant monoclonal antibody recognition ability, such as... Figure 4 As shown in (A).
[0063] Example 4
[0064] Sequencing of monoclonal antibodies
[0065] After expanding the culture of hybridoma cells screened in Example 2, total RNA was extracted and reverse transcribed to synthesize first-strand cDNA. Degenerate primers were designed based on the conserved sequence of the constant region of the mouse antibody for PCR amplification. The amplification results showed that the heavy chain subclass of the monoclonal antibody 1F1 was IgG1 and the light chain subclass was Kappa. Sequencing revealed the amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region of the monoclonal antibody, as shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, and SEQ ID No. 9, respectively; the nucleotide sequences are shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, respectively.
[0066] Example 5
[0067] 5. Identification of B-cell epitopes from monoclonal antibodies
[0068] The overall approach to epitope identification involved progressively constructing a series of truncated fragments of the GAstV Cap protein. Primer sequences for these truncated fragments are shown in Table 1. These fragments were then inserted into the PGEX-4T-1 prokaryotic expression plasmid. The target fragment fused with the GST tag protein to form a recombinant protein. Expression was induced and validated using Western blotting. The results showed successful expression. Figure 2 As shown.
[0069] 5.1 Preliminary identification of monoclonal antibody antigen recognition epitopes
[0070] The full-length GAstV Cap was truncated into six fragments using a binary splitting method (one fragment was divided into two, then two into four). These fragments were then inserted into the PGEX-4T-1 prokaryotic expression plasmid to construct a recombinant fusion protein containing the GST tag. The expression of the truncated GST-tagged fusion protein was detected using an anti-GST antibody. The successfully constructed fusion protein was induced to express and then subjected to Western blotting to preliminarily screen the recognition regions of the monoclonal antibody.
[0071] 5.2 Precise Identification of B-cell Antigenic Epitopes from Monoclonal Antibodies
[0072] The initial positive fragments were further truncated using a dichotomy method. The truncated fragments were then used to construct recombinant fusion proteins containing the GST tag for further screening. This process was repeated until the fragments were truncated to approximately 10 amino acids. Amino acids were then sequentially removed from both ends of the selected positive fragments to construct proteins of varying lengths in a progressively shorter manner. Western blotting was then used for screening until the shortest B-cell antigen epitope was identified. The results are as follows: Figure 5 , Figure 6 As shown, the identified epitope amino acid sequence is shown in SEQ ID No. 10.
[0073] Table 1. Primers used in constructing the GAstV Cap truncated protein
[0074]
[0075]
[0076]
[0077] Example 6
[0078] Monoclonal antibody B-cell antigenic epitope analysis
[0079] The selected GAstV Cap monoclonal antibody B-cell epitopes were compared with the Cap protein sequences of different GAstV strains using BLAST to determine their degree of conservation. The results are as follows: Figure 7 As shown.
[0080] The specification and drawings of this invention are intended to be illustrative rather than restrictive. Based on this invention, those skilled in the art can make substitutions and modifications to some of the technical features without creative effort, and all such modifications are within the scope of protection of this invention.
Claims
1. A monoclonal antibody against type 2 goose astrovirus, characterized in that, The antigen of the monoclonal antibody is the capsid protein of type 2 goose astrovirus. The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, and the amino acid sequences are shown in SEQ ID No. 3 and SEQ ID No. 5, respectively.
2. The type 2 goose astrovirus monoclonal antibody according to claim 1, characterized in that, The heavy chain constant region of the monoclonal antibody is The light chain constant region is of the Kappa type.
3. The type 2 goose astrovirus monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody further includes a heavy chain constant region and a light chain constant region, with amino acid sequences shown in SEQ ID No. 7 and SEQ ID No. 9, respectively.
4. The use of the monoclonal antibody against type 2 goose astrovirus as described in any one of claims 1-3 in the preparation of serological diagnostic reagents for type 2 goose astrovirus.