An antigen epitope peptide and a paralichthys olivaceus mucin muc13 specific antibody prepared therefrom

Abstract

The application provides an antigen epitope peptide and a paralichthys olivaceus mucin Muc13 specific antibody prepared by the antigen epitope peptide, determines a B cell epitope from the paralichthys olivaceus Muc13 protein through antigen epitope screening, and the sequence is PGTTEEPDPTAGSPP. After the epitope is coupled with a KLH carrier protein, the epitope is used for immunizing New Zealand white rabbits, and a rabbit-derived anti-paralichthys olivaceus Muc13 specific antibody is successfully obtained. The antibody can specifically recognize the Muc13 protein in paralichthys olivaceus mucosa tissue and Muc13 positive mucous cells, and provides a key detection means for in-depth exploration of the function mechanism of mucous cells in the fish mucosa immune response process.

Description

Technical Field

[0001] This invention belongs to the field of fish immune products technology, specifically relating to an antigenic epitope peptide and its preparation in turbot ( Paralichthys olivaceus Mucin Muc13 specific antibody. Background Technology

[0002] Turbot is an important farmed fish species in Northeast Asia; however, high-density farming methods lead to frequent disease outbreaks, severely hindering the healthy development of the industry. The mucus barrier, as the first line of defense against pathogen invasion in fish, directly determines fish health and survival rates. Currently, there is a lack of specific molecular tools for evaluating the mucus immune function of fish, making it difficult to accurately monitor the state of the mucus barrier.

[0003] Over a long period of evolution, fish have developed a unique mucosal immune system to cope with the challenges of complex aquatic environments. As a key component of this immune system, goblet cells (also known as mucus cells) are abundant in mucosal tissues such as gills, intestines, and skin. These cells continuously secrete mucus, forming a dynamically renewing gel-like barrier on the mucosal surface. This mucus layer is not only the first line of physical defense but also a highly dynamic, immune-active interface: its continuous secretion and shedding mechanisms effectively flush the mucosal surface, hindering the colonization and retention of pathogenic microorganisms. Simultaneously, the mucus layer acts as a medium connecting the internal and external aquatic environment, participating in various physiological processes such as osmotic pressure regulation, gas exchange, and ion balance maintenance. Given that fish live their entire lives in a microbial-rich aquatic environment, the integrity and functional state of their mucosal surface mucus barrier directly affect their health and resistance to pathogen infection, holding an irreplaceable core position in the fish's immune defense system.

[0004] Mucins, as core structural components of mucogel networks, can be divided into two main categories based on their molecular structure and binding mode to the cell membrane: secretory mucins and membrane-bound mucins. Membrane-bound mucins anchor to the apical membrane of epithelial cells through their transmembrane domains, directly participating in biological processes such as mucosal barrier maintenance, cell signal transduction, and pathogen recognition. Muc13, a typical representative of the membrane-bound mucin family, plays a crucial role in maintaining mucosal epithelial homeostasis. In recent years, significant progress has been made in functional studies of Muc13 in mammals. Studies have shown that Muc13 shifts from its normal apical membrane localization to abnormally high cytoplasmic expression in colorectal cancer tissues, promoting tumor cell survival and chemotherapy resistance by activating the NF-κB signaling pathway. Simultaneously, Muc13, as a direct transcriptional target of RUNX1, participates in the activation of the Wnt / β-catenin pathway, driving tumor metastasis. In ovarian cancer, Muc13 also exhibits significantly high expression, making it an important target for antibody-drug conjugate development. These findings not only reveal the multiple regulatory roles of Muc13 in disease development and progression but also make it an important molecular target for disease diagnosis and treatment. However, compared to in-depth research in mammals, research on Muc13 in fish is still in its early stages. Current understanding of fish Muc13 is mainly limited to genome-level sequence annotation; its tissue distribution, cellular localization, and dynamic changes during immune responses at the protein level remain unclear, and in-depth analysis of its functional mechanisms is lacking. A key obstacle to this research status is the lack of specific detection tools. Due to the absence of specific antibodies capable of recognizing fish Muc13 protein, precise localization and quantification of fish Muc13 at the protein level are difficult, severely hindering the research progress on fish mucosal immune defense mechanisms. Therefore, developing highly efficient and specific fish Muc13 antibodies is of significant theoretical and practical value for a deeper understanding of the molecule's function in fish mucosal immunity and for exploring its role in disease development. Summary of the Invention

[0005] This invention provides an antigenic epitope peptide and a specific antibody against Muc13 mucin in turbot prepared therefrom. The prepared antibody can specifically bind to Muc13 mucin in turbot and effectively trace Muc13-positive mucus cells in mucosal tissue. This makes it possible to explore the function of mucus cells in the mucosal immune defense of turbot and to elucidate the immune regulatory network of mucin, which is of great significance for aquatic animal immunology research and disease control.

[0006] This invention first provides a B-cell antigenic epitope polypeptide for preparing a Muc13-specific antibody against turbot, wherein the epitope polypeptide comprises:

[0007] 1) A polypeptide with the amino acid sequence PGTTEEPDPTAGSPP (SEQ ID NO:1);

[0008] 2) A polypeptide obtained by substituting, deleting, or adding one or more amino acids in 1), and having the efficacy of preparing a turbot Muc13-specific antibody against the polypeptide described in 1).

[0009] 3) A molecule for enhancing immune effects is coupled to the polypeptide with the amino acid sequence PGTTEEPDPTAGSPP (SEQ ID NO:1);

[0010] As a specific example, the molecule used to enhance the immune effect is hemocyanin;

[0011] This invention also provides an application of the aforementioned antigenic epitope polypeptide in the preparation of antibodies;

[0012] The antibody described herein, as a specific example, is a monoclonal antibody.

[0013] The present invention also provides a specific antibody, which is prepared by immunizing animals with the above-mentioned antigenic polypeptide;

[0014] The animal described, as a specific example, is the New Zealand White Rabbit;

[0015] The method for preparing the specific antibody in this invention involves conjugating the B-cell antigen epitope of turbot Muc13 to a vector, immunizing New Zealand white rabbits, and then processing the serum to obtain the anti-turbot Muc13 specific antibody.

[0016] The carrier described herein, as a specific example, is a KLH (hemocyanin) carrier;

[0017] The application of the specific antibody against turbot Muc13 provided by this invention in the preparation of a reagent for identifying turbot mucin Muc13.

[0018] The application of the specific antibody against turbot Muc13 provided by this invention in the preparation of reagents for studying Muc13-positive mucin cell immune responses.

[0019] The present invention also provides a kit for detecting or tracing turbot mucin Muc13 and Muc13-positive mucus cells in paraffin or frozen sections, the kit containing the above-mentioned specific antibody.

[0020] The antigenic epitope peptide provided by this invention can be used to prepare specific antibodies against the turbot mucin Muc13. Verification results show that the prepared antibody can specifically recognize Muc13 protein and Muc13-positive mucus cells in turbot mucosal tissue, achieving in-situ labeling and localization of target cells. Using this antibody, the expression dynamics of Muc13 in turbot under pathogen infection or immunization conditions can be detected at both the gene and protein expression levels, providing important technical support for elucidating the functional mechanism of mucin in the fish mucosal immune response. Simultaneously, this antibody, as a high-affinity molecular probe targeting Muc13, shows broad application potential in the immunodetection of diseases related to turbot mucus secretion disorders and the development of related diagnostic reagents. Attached Figure Description

[0021] Figure 1 Phylogenetic tree of Muc13 molecules constructed using the neighbor-joining method;

[0022] Figure 2 Transmembrane sequence analysis of Muc13 in turbot;

[0023] Figure 3 : Sequence analysis diagram of Muc13 signal peptide in turbot;

[0024] Figure 4 : Analysis diagram of the structural domains of the Muc13 protein in turbot;

[0025] Figure 5 : Exon and intron distribution diagram of the turbot Muc13 molecule;

[0026] Figure 6 Secondary structure diagram of turbot Muc13;

[0027] Figure 7 : Prediction map of linear antigenic epitopes of Muc13 on B cells analyzed by IEDB software;

[0028] Figure 8 : Analysis of antigenic epitope parameters of the Muc13 molecule predicted by DNAStar software;

[0029] Figure 9 : A schematic diagram of the tertiary structure model of the Muc13 protein molecule and the finally selected antigen peptide.

[0030] Figure 10 Western blot analysis of the binding of Muc13-specific antibody to native tissue proteins of turbot intestine, where M represents marker and NC represents negative control.

[0031] Figure 11Image showing the specific binding reaction of Muc13 antibody to the mucin Muc13 in the gills, hindgut, skin, and olfactory sac of turbot using indirect immunofluorescence assay (40× objective). Neg (Negetive) indicates the negative control. Detailed Implementation

[0032] This invention discloses a method for preparing a Muc13 antibody against turbot and its application. By analyzing the antigenic characteristics of the Muc13 molecule using bioinformatics methods, dominant antigenic epitopes were screened and obtained. Based on this, a specific antibody with high recognition activity was prepared. This antibody can effectively label Muc13-positive mucus cells in turbot mucosal tissue and Muc13 in mucus on the mucosal surface, laying an important technical foundation for exploring the physiological function of mucus cells, assessing the immune response status of fish, and conducting research on related disease prevention and control.

[0033] Indirect immunofluorescence labeling of turbot tissue sections was performed using the Muc13 antibody prepared according to this invention. The results showed that specific positive signals were detected in the gills, hindgut, skin, and olfactory sac. The signal was clearly distributed on the cell membrane of mucus cells, appearing as punctate and circular patterns.

[0034] A series of validation experiments on antibody performance showed that: indirect ELISA detection confirmed that the prepared antibody could specifically bind to the Muc13 antigen peptide; Western blot analysis showed that the antibody could specifically recognize the natural mucin in the intestinal tissue of turbot, while the negative rabbit serum control showed no binding signal, indicating that the prepared antibody has good specificity; immunofluorescence combined with WGA histochemical detection further confirmed that the Muc13 antibody could accurately recognize WGA-positive mucin cells in the gills, hindgut, skin, and olfactory sac tissue of turbot, achieving in situ visualization of target cells. The invention will now be described in detail with reference to embodiments and accompanying drawings.

[0035] Example 1: Gene cloning of the turbot Muc13 molecule

[0036] 1) Primer design for conserved gene sequences

[0037] A systematic bioinformatics analysis of the Muc13 gene from the turbot was performed. Gene structure analysis revealed that the gene consists of 12 exons and 11 introns. Domain prediction results showed that the encoded protein contains characteristic structural modules of membrane-bound mucins, namely the epidermal growth factor-like domain (EGF domain), the SEA domain (sea urchin spermin-enterokinase-aggregin domain), and the RPT1 internal repeat sequence domain. Analysis using SignalP and TMHMM online tools confirmed that amino acid residues 1-19 at the N-terminus of the protein constitute the signal peptide sequence, and that it possesses typical transmembrane and intracellular regions, further confirming that Muc13 belongs to the membrane-bound mucin family. Phylogenetic tree construction based on the neighbor-joining method showed that Muc13 in bony fishes clustered into an independent branch, and its clustering pattern was highly consistent with the species' evolutionary history. Among them, the turbot Muc13 and the European yellowfin flounder (… Limanda limanda They are most closely related by blood.

[0038] Using the CDS sequence of the turbot Muc13 gene published in the NCBI database as a template, specific amplification primers were designed using Primer Premier 5.0 software. During the design process, key indicators such as the primers' secondary structure forming ability (including dimers and hairpin structures) and annealing temperature were evaluated and optimized. The final primer sequences are: forward primer 5'-CTGGGAAATACAACGTGTTTTTCGT-3', and reverse primer 5'-TTTTCCATCGTTTTGCGTGTAAGGA-3'. These primers were synthesized by Qingke Biotechnology Co., Ltd.

[0039] 2) Amplification of the gene core fragment

[0040] Using cDNA synthesized from total RNA reverse transcribed from turbot tissue as a template, PCR amplification was performed using specific primers designed for the CDS region of the Muc13 gene. After amplification, the reaction product was separated by electrophoresis on a 1.0% agarose gel. The target band, matching the expected molecular weight, was observed and excised using a gel imaging system. After gel purification, the band was ligated into the pMD19-T vector and transformed into *E. coli* DH5α competent cells. Positive single colonies were selected by colony PCR screening and sent to Beijing Qingke Biotechnology Co., Ltd. for bidirectional sequencing. BLAST alignment analysis of the obtained sequence with known sequences in the NCBI database confirmed that the length of the amplified product was 1857 bp, completely consistent with the expected sequence.

[0041] (3) Bioinformatics analysis of the Muc13 protein sequence of turbot

[0042] BLAST results showed that the Muc13 protein possesses typical EGF-like domains, SEA domains, and RPT1 domains characteristic of membrane-bound mucins. A phylogenetic tree of the turbot Muc13 molecule was constructed using MEGA 5.0 software. Figure 1 Multiple sequence alignment analysis of the obtained turbot Muc13 protein sequence with sequences from other species was performed using DNAMAN software. The results showed that the turbot Muc13 molecule is similar to that of the European yellowfin flounder (…). Limanda limanda Muc13 has the highest sequence homology.

[0043] Example 2: Screening of Muc13 antigenic peptide epitopes in turbot

[0044] (1) The transmembrane structure of the turbot Muc13 protein was predicted using the TMHMM online analysis tool (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ). The results showed that the protein has typical transmembrane domains and intracellular regions, consistent with the structural characteristics of membrane-bound mucins. Figure 2 Meanwhile, the SignalP 5.0 server (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) was used to predict the signal peptide sequence of the Muc13 protein. The analysis results showed that amino acid residues 1-19 at the N-terminus of this protein constitute the signal peptide sequence. Figure 3 ).

[0045] (2) To analyze the structural features of the Muc13 protein, its amino acid sequence was functionally identified using an online prediction tool. The results showed that the protein contains several characteristic structural units: a typical EGF domain (positions 224-262), a SEA domain (positions 265-369), an EGF-like domain (positions 371-413), and a transmembrane domain (positions 448-470). Furthermore, an RPT1 domain was identified at the C-terminal region of the protein. This domain is formed by the folding of two discontinuous amino acid sequences at positions 576-594 and 596-614. Figure 4 ).

[0046] (3) The number of exons and introns in the Muc13 gene of turbot are 12 and 11, respectively. Figure 5 ).

[0047] (4) Analysis of the secondary structure of turbot Muc13 protein showed that the β-sheet and random coil sections exhibited good immunogenicity and reactivity; α-helices accounted for 11.5%, extended chains for 14.25%, and random coils for 74.25%. The distribution of various structures in the amino acid sequence of turbot Muc13 is shown in [Figure number missing]. Figure 6 .

[0048] (5) Analysis of the antigens of the Muc13 protein revealed that the main B-cell linear antigenic sites of the Muc13 protein are amino acid residues 35-239, 373-394, 472-486, and 488-540. Figure 7 ).

[0049] (6) The amino acid sequence of the turbot Muc13 protein was comprehensively analyzed using DNAStar bioinformatics software to systematically evaluate its hydrophilicity (Kyte-Doolittle protocol), flexibility (Karplus-Schulz protocol), antigenicity (Jameson-Wolf protocol), and surface accessibility (Emini protocol). Based on these parameters, using β-turn and random coil regions as the screening scope, amino acid segments that simultaneously meet the criteria of hydrophilicity index ≥ 0, surface accessibility index ≥ 1, and antigenicity index ≥ 0 were selected as candidate antigenic epitopes. The analysis results showed that multiple potential antigenic epitopes meeting the above screening criteria exist in the turbot Muc13 molecule. Figure 8 ).

[0050] The three-dimensional spatial conformation of the turbot Muc13 protein was predicted and modeled using the AlphaFold3 deep learning system. The predicted protein structure model was rendered and optimized using PyMOL molecular visualization software, and then mapped and compared with known crystal structure models to highlight the structural features of the target protein. To further verify the accessibility of candidate epitopes, the screened epitopes were highlighted as chromospheres in the three-dimensional structural model. The results showed that all candidate epitopes were located on the outer surface of the protein molecule, exhibiting good solvent accessibility and possessing the spatial conformational basis for specific antibody recognition. Figure 9 ).

[0051] Example 3: Preparation of Muc13-specific antibody against turbot

[0052] 1) Determination of antigenic peptide sites

[0053] Based on the prediction results of Example 2, a multi-level screening strategy was used to identify the dominant antigenic epitopes of the Muc13 protein: after eliminating epitopes located in the signal peptide, transmembrane region, and intracellular region, potential B-cell linear epitopes were screened by combining antigen parameter analysis and secondary structural features (random coils, β-turn regions). Further spatial localization verification was performed using a three-dimensional structural model to confirm the candidate antigenic peptide sequences. 64 PGTTEEPDPTAGSPP 78 Located on the surface of protein molecules, it possesses the spatial conformational basis for antibody recognition, making it suitable as an immunogen for Muc13-specific antibodies. The antigenic peptide was synthesized in vitro and conjugated to hemocyanin; mass spectrometry analysis showed that its purity met the requirements for subsequent immunization.

[0054] (2) Immunity

[0055] A specific polyclonal antibody was prepared by immunizing New Zealand white rabbits with a Muc13 antigenic peptide-hemocyanin (KLH) conjugate complex. The immunization program consisted of a primary immunization and four booster immunizations, for a total of five immunizations, with an interval of 7 days between immunizations. For the primary immunization, the immunogen was thoroughly emulsified with an equal volume of Freund's complete adjuvant; for the booster immunizations, an equal volume of Freund's incomplete adjuvant was used for emulsification. The emulsified immunogen was injected subcutaneously at six injection sites on the rabbit's back and groin, with 100 μL injected at each site. Three days after the last immunization, whole blood was collected via cardiac sampling. The collected blood was allowed to stand at room temperature for 3 hours, then transferred to a 4°C freezer overnight. The next day, the blood sample was centrifuged at 8000 g for 15 minutes at 4°C, and the supernatant was carefully aspirated, aliquoted, and frozen at -80°C to obtain the rabbit anti-Muc13 specific polyclonal antibody.

[0056] Example 4: ELISA determination of Muc13 antibody titer

[0057] (1) Adjust the antigen concentration to 50 μg / mL with PBS, add 100 μL to each well of a 96-well plate, and perform 3 replicates. The negative control is rabbit negative serum, and the positive control is encapsulated with irrelevant proteins and incubated with the uncoupled KLH Muc13 antigen peptide.

[0058] (2) Coat overnight at 4 ℃. The next day, discard the coating solution, add 100 μL PBST to the plate, shake and wash, repeat 3 times, 5 min each time. Finally, wipe the plate clean on filter paper, add 100 μL BSA solution, and block at 37 ℃ for 1.5 h.

[0059] (3) Repeat the washing process above, add 100 μL of specific antibody to each well, and incubate at 37 °C for 1 h.

[0060] (4) Wash three times with PBST, spin dry, add 100 μL of alkaline phosphatase-labeled secondary antibody, incubate at 37 °C for 1 h, and wash again.

[0061] (5) Add 100 μL of pNPP color development solution, develop color in the dark for 10 min, preheat the microplate reader for 30 min and then detect OD 405, 20 cycles.

[0062] The result showed that the titer of Muc13 antibody was 1:12000.

[0063] Example 5: Identification of Muc13-specific antibodies by Western blot

[0064] (1) SDS-AGE electrophoresis

[0065] ① The previously extracted and purified turbot intestinal tissue protein was added to a sample buffer containing sodium dodecyl sulfate in equal proportions, reduced at 100 °C for 10 min, and then iodoacetamide with a final concentration of 25 mM was added. The mixture was then incubated at room temperature in the dark for 1 h.

[0066] ② Add the sample treated in ① to each well, 10 µL of sample per well. Electrophoresis is performed at 100 V for 1 h until the blue band indicated by bromophenol blue reaches 2 / 3 of the gel. Remove the gel for transfer to a membrane.

[0067] ③ Cut a polyvinylidene fluoride (PVDF) membrane (0.45 μm pore size) the same size as the electrophoresis gel, activate it by soaking in methanol, and then transfer it to electrotransfer buffer (25 mmol / L Tris-Base, 192 mmol / L glycine, pH 8.3) for equilibration. Simultaneously, briefly equilibrate the electrophoretically extracted gel in the electrotransfer buffer. Prepare the transfer interlayer in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge," carefully removing air bubbles between each layer.

[0068] ④ Place the assembled transfer clamp into the electrotransfer tank, with the gel facing the negative electrode and the membrane facing the positive electrode. Set the electrophoresis constant current to 200 mA and perform protein electrotransfer for 5 h.

[0069] ⑤ After the transfer is complete, remove the PVDF membrane.

[0070] (2) Immunoblotting:

[0071] ① Wash the PVDF membrane with PBS for 15 min, then block it in 5% BSA for 1 h at 37 ℃;

[0072] ② Wash three times with PBST, 5 min each time; add turbot Muc13 antibody and incubate at 37 ℃ for 1 h. Use rabbit negative serum as a negative control;

[0073] ③ Wash three times using the same method as ②;

[0074] ④ Place the PVDF membrane in a horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody solution (diluted to 1:50000) with appropriate amount of blocking solution or TBST, and incubate slowly with shaking in a constant temperature shaker at 37 ℃ for 1 h.

[0075] ⑤ Wash three times using the same method as in ②;

[0076] ⑥ Immerse the PVDF membrane in freshly prepared enhanced chemiluminescence substrate working solution and incubate at room temperature in the dark for 1-2 minutes. After incubation, gently drain excess liquid from the membrane surface and wrap the membrane flat with transparent plastic wrap. Then place the wrapped PVDF membrane in a chemiluminescence imaging system to acquire chemiluminescence signal images of the target protein.

[0077] The recognition ability of the prepared rabbit anti-turbot Muc13 specific antibody was evaluated using Western blot. Using total protein from turbot intestinal tissue as the detection sample, the results showed that the prepared antibody detected a single specific band at approximately 130 kDa, while no color signal was observed at this position in the negative control serum. Figure 10 This indicates that the prepared antibody can specifically recognize the natural Muc13 protein in turbot intestine tissue.

[0078] Example 5: Indirect immunofluorescence identification of turbot Muc13 antibody

[0079] ① Healthy turbot (body weight 60±5 g) were selected as experimental subjects and tissue samples were collected after one week of temporary rearing. Gill, hindgut, skin, and olfactory sac tissues were collected and rinsed in 0.01 M phosphate-buffered saline (PBS, pH 7.4) to remove surface deposits. After absorbing residual moisture with qualitative filter paper, the tissues were gently picked up with forceps and placed in a square foil container pre-filled with OCT embedding medium. The tissue orientation was adjusted using a dissecting needle to ensure proper embedding and sectioning. The embedding container was then quickly transferred to a -80 ℃ freezer for rapid freezing and storage, ready for subsequent sectioning.

[0080] ② Fix the embedded tissue block onto the sample holder of the cryostat, set the section thickness to 6 μm, and cut 5 tissue sections consecutively. Gently attach the sections to an adhesive slide and pick them up. Immediately immerse the sections in pre-cooled acetone at 4 ℃ and fix for 15 min. After fixation, remove the sections and place them in a fume hood to dry thoroughly. After the acetone has completely evaporated, transfer them to a -20 ℃ freezer and seal for later use.

[0081] ③ Remove the frozen sections from -20℃ and allow them to warm to room temperature for 15 min. Wash with PBST three times on a shaker for 5 min each time. After drying, circle the tissue area with an immunohistochemical pen, add 5% BSA (prepared with 0.01 M PBS) blocking solution, and incubate in a humidified chamber at 37℃ for 1 h.

[0082] ④ Discard the blocking solution on the surface of the slide and shake dry slightly. Add the primary antibody working solution to the tissue area, i.e., rabbit anti-turbot Muc13 serum diluted 1:1000 with PBST buffer (PBS containing 0.05% Tween-20), ensuring that the antibody solution completely covers the entire tissue slide. Place the slide stably in a humidified chamber and incubate at 37 ℃ for 1 h. After incubation, discard the primary antibody solution, place the slide on a shaker, and wash with PBST buffer three times for 5 min each time to thoroughly remove unbound primary antibody and non-specific adsorption.

[0083] ⑤ After incubation with the primary antibody and washing, place the slides in a centrifuge to remove residual liquid from the slide surface. Add the mixed working solution to the tissue area. This working solution contains Cy3-labeled goat anti-rabbit IgG fluorescent secondary antibody diluted 1:1000 with PBST, and FITC-labeled wheat germ lectin (FITC-WGA) diluted 1:1000 with PBS. The latter is used to stain mucin. After ensuring that the mixture evenly covers the entire tissue slide, place the slides stably in a humidified chamber and incubate at 37°C in the dark for 45 min.

[0084] ⑥ Remove the slide and wash it three times with PBST for 5 minutes each time. Add DAPI (1:2000 dilution), incubate at room temperature for 15 minutes, and then wash as above.

[0085] ⑦ After drying, add anti-fluorescence quenching mounting medium in the dark, and then cover with a coverslip.

[0086] ⑧ Results observed under a fluorescence microscope.

[0087] The tissue-level recognition ability of the prepared rabbit anti-turbot Muc13 antibody was detected using indirect immunofluorescence. The results showed that the antibody could specifically label the natural mucin in the gills, hindgut, skin, and olfactory sac tissues of turbot, exhibiting a clear positive fluorescent signal. Figure 11The fluorescent signal was mainly localized in the cell membrane region of mucin cells, exhibiting a ring-like or granular aggregation distribution, with some signals also observed at the apex of epithelial cells. Using FITC-WGA-counted rabbit negative serum as a negative control, no non-specific fluorescent signal of the Cy3 channel was observed in tissue sections, confirming the reliability of the antibody's positive signal. To further verify the antibody's recognition specificity, co-localization analysis was performed using immunofluorescence and WGA co-staining techniques. WGA, as a phytohemagglutinin that specifically binds to the glycosylation sites of mucins, is a commonly used probe for labeling mucin substances. The co-localization results showed that the immunofluorescence signal of the Muc13 antibody and the WGA staining signal highly overlapped on the mucin cell membrane, further confirming the antibody's specific recognition ability of the membrane-bound mucin Muc13.

[0088] In summary, this invention successfully prepared a specific antibody against Muc13, a membrane-bound mucin found in turbot. This antibody can serve as a molecular probe for recognizing mucus cells and mucins, detecting changes in Muc13 expression after pathogen infection or immunization, and assessing the immune status of mucosal tissues. The development of this antibody provides a crucial detection tool for fish mucosal immunity research, offers quantitative indicators for evaluating the immunogenicity of novel mucosal preparations, and lays the foundation for establishing health monitoring technologies for farmed fish based on mucus barrier function and developing drugs that regulate mucus. It also has broad application prospects in the precise control of diseases in turbot aquaculture.

[0089] Those skilled in the art will understand that modifications, additions, and substitutions to the above embodiments are possible within the scope of protection of this invention, and none of them exceed the scope of protection claimed by this invention.

Claims

1. An antigenic epitope peptide, characterized in that, The epitope peptide comprises: 1) A polypeptide with the amino acid sequence SEQ ID NO:1; 2) A polypeptide obtained by substituting, deleting, or adding one or more amino acids in 1), and having the efficacy of preparing a turbot Muc13-specific antibody against the polypeptide described in 1). 3) A polypeptide prepared by coupling a molecule for enhancing immune effects to a polypeptide with the amino acid sequence SEQ ID NO:

1.

2. The antigenic epitope peptide according to claim 1, characterized in that, The molecule that enhances the immune effect is hemocyanin.

3. The use of the antigenic epitope peptide according to claim 1 in the preparation of antibodies.

4. The application as described in claim 3, characterized in that, The antibody mentioned is a monoclonal antibody.

5. A specific antibody, characterized in that, The specific antibody is prepared by immunizing animals with the antigenic epitope peptide described in claim 1.

6. The specific antibody as described in claim 5, characterized in that, The animal in question is a New Zealand white rabbit.

7. The specific antibody as described in claim 5, characterized in that, The specific antibody described herein is prepared by conjugating the antigenic epitope peptide described in claim 1 to a carrier, immunizing New Zealand white rabbits, and then processing the serum to obtain a specific antibody against turbot Muc13.

8. The specific antibody as described in claim 7, characterized in that, The carrier is a hemocyanin carrier.

9. The use of the specific antibody according to claim 5 in the preparation of a reagent for identifying mucin Muc13 in turbot.

10. A kit for detecting or tracing turbot mucin Muc13 and Muc13-positive mucus cells in paraffin or frozen sections, characterized in that, The kit contains the specific antibody as described in claim 5.

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