An antigenic epitope polypeptide and a paralichthys olivaceus mucin Muc1 specific antibody prepared therefrom

Abstract

The application provides an antigen epitope polypeptide and a paralichthys ovatus mucin Muc1 specific antibody prepared by the antigen epitope, a B cell epitope from the paralichthys ovatus Muc1 protein is identified through antigen epitope screening, and the amino acid sequence is SEQ ID NO:1. After the epitope is coupled with a KLH carrier protein, a New Zealand white rabbit is immunized, and a rabbit anti-paralichthys ovatus Muc1 polyclonal antibody is successfully prepared. The antibody can specifically recognize the Muc1 protein in paralichthys ovatus mucosa tissue and positive mucous cells, and provides an important detection tool for studying the function mechanism of mucous cells in fish mucosal immunity.

Description

Technical Field

[0001] This invention belongs to the field of fish immune labeling technology, specifically relating to an antigenic epitope polypeptide and its preparation in turbot ( Paralichthys olivaceus Mucin Muc1 specific antibody. Background Technology

[0002] Over a long period of evolution, fish have gradually developed a mucosal immune defense system adapted to the characteristics of their aquatic environment, enabling them to cope with the constant pressure from various pathogenic microorganisms in the water. Mucosal tissues such as the gills, intestines, skin, and nasal cavity are important components of this immune system. Numerous goblet cells are widely distributed in the epithelial layer of these tissues, capable of synthesizing and secreting mucus. The secreted mucus covers the mucosal surface, forming a continuously renewing gel-like barrier structure. This mucus layer not only serves as a physical barrier but also acts as an immunologically active functional interface. On one hand, the continuous secretion and natural shedding of mucus create a dynamically renewed flushing mechanism, reducing the adhesion and colonization of exogenous pathogenic microorganisms on the mucosal surface. On the other hand, this interface also participates in various important physiological processes in fish, such as osmotic regulation and gas exchange. Therefore, under the long-term exposure to a complex microbial environment, the structural integrity and immunomodulatory function of the mucosal mucus barrier are crucial for the host's resistance to pathogen infection and are an important component of the fish's immune defense system.

[0003] Muc1 is a transmembrane glycoprotein with typical structural features. Its molecules typically consist of three functional regions: a highly glycosylated extracellular domain, a single-transmembrane domain, and an intracellular tail with signal transduction functions. This structure enables it to simultaneously recognize external signals and transmit intracellular signals. Its extracellular region is rich in glycosylation sites, which can serve as microbial binding sites, thus hindering pathogen adhesion to epithelial cell surfaces to some extent. Simultaneously, the intracellular tail contains multiple phosphorylated sites, which can recruit related signaling molecules to participate in the regulation of cellular signaling pathways, thereby influencing inflammatory and immune responses. As an important member of the membrane-bound mucin family, Muc1 has been extensively studied in mammals. Existing research has shown that abnormal Muc1 expression is closely related to the development and progression of various diseases. For example, in colorectal cancer tissues, Muc1 changes from its normal apical membrane localization to abnormal cytoplasmic expression and can enhance chemotherapy resistance in tumor cells by activating the NF-κB signaling pathway. Furthermore, Muc1 can also participate in the expression regulation of tumor metastasis-related genes as an upstream regulator of the Wnt / β-catenin signaling pathway. Muc1 exhibits significantly high expression in various tumor tissues, including ovarian cancer, pancreatic cancer, and non-small cell lung cancer, thus gradually becoming an important research target for antibody-targeted therapy and CAR-T cell immunotherapy. Currently, several immunotherapy strategies targeting Muc1 have entered the clinical research stage, including monoclonal antibodies, tumor vaccines, and CAR-T cell therapy.

[0004] However, compared to mammals, research on Muc1 in fish remains relatively limited. Existing studies mainly focus on sequence identification at the genomic level. For example, the Muc1 gene has been reported in economically important fish such as carp and Atlantic salmon, and it belongs to the mucin family. However, systematic experimental data on the expression distribution, cellular localization characteristics, expression changes, secretion patterns, and regulatory mechanisms of fish Muc1 protein in different tissues, under pathogen infection or immune stimulation conditions, are still lacking. This insufficient research foundation limits the in-depth analysis of the mucosal immune mechanisms in fish. Therefore, developing specific detection tools for fish Muc1, especially preparing specific antibodies, is of great significance for conducting related functional studies.

[0005] Turbot is one of the important marine aquaculture species in the coastal areas of northern my country. Under the conditions of high-density factory farming, bacterial and viral diseases occur frequently, causing significant economic losses to the aquaculture industry. Most pathogenic microorganisms enter the fish body mainly through the mucosal surfaces such as gills, skin, and intestines; therefore, the structural integrity and functional status of the mucosal barrier are closely related to the health of fish. In recent years, mucosal immunization has gradually become an important direction in fish vaccine research and development. Among them, mucosal inoculation methods such as immersion immunization and oral immunization have received widespread attention due to their advantages of simple operation and minimal stress response. However, whether the vaccine antigen can effectively cross the mucus barrier and enter the epithelial tissue is an important factor affecting the immune response. Therefore, it is necessary to further study the structural composition and functional characteristics of the mucus barrier. However, the lack of specific detection tools for mucins currently limits the development of related research. Summary of the Invention

[0006] This invention provides an antigenic epitope polypeptide and its preparation in turbot ( Paralichthys olivaceus A specific antibody against the mucin Muc1 was developed. This antibody can specifically recognize and trace the Muc1 protein and Muc1-positive mucus cells in turbot, solving the problem of the lack of effective detection tools in turbot mucosal immunity research. This achievement lays an important foundation for further elucidating the function of mucus cells in mucosal immune defense and clarifying the immune regulatory network of mucins. It has significant scientific and application value for improving turbot disease control and enriching fish immunology theory.

[0007] This invention first provides a turbot B-cell antigenic epitope polypeptide, which comprises:

[0008] 1) A polypeptide with the amino acid sequence TTEPKPKPTSSTTEQ (SEQ ID NO:1);

[0009] 2) Peptides obtained by substituting, deleting, or adding one or more amino acids to the polypeptide in 1);

[0010] The present invention also provides a coupled protein, wherein a molecule for enhancing immune effects is coupled to the 5′ or 3′ end of a B cell antigenic epitope polypeptide with the amino acid sequence SEQ ID NO:1.

[0011] Furthermore, the molecule used to enhance immune effects is hemocyanin.

[0012] The present invention also provides an application of the aforementioned turbot B cell antigenic epitope polypeptide in the preparation of antibodies.

[0013] In another aspect, the present invention provides a specific antibody prepared by immunizing animals with the above-mentioned turbot B cell antigenic epitope polypeptide as an antigen.

[0014] The animal described, as a specific example, is the New Zealand white.

[0015] The method for preparing the specific antibody in this invention involves conjugating the B-cell antigen epitope of turbot Muc1 to a vector, immunizing New Zealand white rabbits, and then processing the serum to obtain the anti-turbot Muc1 specific antibody.

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

[0017] The application of the specific antibody against the turbot Muc1 molecule provided in this invention in the preparation of a reagent for identifying turbot mucin Muc1.

[0018] The application of the specific antibody against the turbot Muc1 molecule provided by this invention in the preparation of reagents for studying Muc1-positive mucin cell immune responses.

[0019] This invention also provides a kit for detecting or tracing turbot mucin Muc1 and Muc1-positive mucus cells in paraffin or frozen sections, the kit comprising:

[0020] 1) Antigen retrieval solution (0.01 M sodium citrate buffer, pH 6.0) is used to break the cross-links between the aldehyde fixative and the antigenic determinant under high temperature conditions, thereby exposing the antigenic epitope;

[0021] 2) 5% bovine serum albumin (BSA) blocking solution (prepared with 0.01 M PBS) is used to block non-specific binding sites on the slides and reduce non-specific adsorption;

[0022] 3) Specific antibodies against turbot Muc1 molecules (1:1000, diluted in 0.01 M PBS) are used to recognize the mucin Muc1 and its positive cells in turbot mucosal tissue;

[0023] 4) Cy3-labeled goat anti-rabbit IgG antibody (1:1000, diluted in 0.01 M PBS) was used to specifically recognize Muc1-specific antibodies, showing Muc1-positive cells;

[0024] 5) DAPI (1:2000, diluted in 0.01 M PBS) was used to stain cell nuclei.

[0025] The 0.01M sodium citrate buffer (pH 6.0) contains the following components: 3.8 mL of 0.1M citric acid solution and 16.2 mL of 0.1M sodium citrate buffer are mixed and then distilled water is added to a final volume of 200 mL.

[0026] The 0.01 M PBS (1 L, pH 7.4) contained the following components: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4, with the pH adjusted to 7.4 using HCl.

[0027] This invention discloses a specific antibody against the mucin Muc1 of turbot, its preparation method, and its applications. Based on the turbot Muc1 gene sequence, the secondary structure, hydrophilicity, and antigenic index of its encoded protein are comprehensively analyzed using bioinformatics techniques to screen for dominant B-cell antigenic epitopes. Based on these epitopes, an antigenic polypeptide is synthesized and conjugated with a carrier protein to immunize experimental animals. After purification, a rabbit-derived anti-turbot Muc1 polyclonal antibody is obtained. Immunohistochemistry and Western blot verification show that this antibody can specifically recognize the Muc1 protein in turbot mucosal tissue and can precisely locate Muc1-positive mucus cells, achieving in-situ visualization of the target cells. Using this antibody, changes in Muc1 expression in turbot during pathogen infection or vaccine immunization can be systematically monitored at the gene and protein levels, providing key technical support for further elucidating the regulatory mechanism of mucus molecules in fish mucosal immune responses. Furthermore, this antibody, as a specific biological probe for recognizing Muc1, has potential application value in the immunological detection and diagnostic reagent development of diseases related to abnormal turbot mucus secretion. Attached Figure Description

[0028] Figure 1 Phylogenetic tree of the Muc1 molecule constructed using the neighbor-joining method;

[0029] Figure 2 Transmembrane sequence analysis of Muc1 in turbot;

[0030] Figure 3 : Sequence analysis diagram of Muc1 signal peptide in turbot;

[0031] Figure 4 : Analysis diagram of the structural domains of the Muc1 protein in turbot;

[0032] Figure 5 : Exon and intron distribution diagram of the Muc1 molecule of turbot;

[0033] Figure 6 Secondary structure diagram of turbot Muc1;

[0034] Figure 7 : Prediction map of B-cell linear antigenic epitopes of Muc1 analyzed by IEDB software;

[0035] Figure 8 : Analysis of antigenic epitope parameters of the Muc1 molecule predicted by DNAStar software;

[0036] Figure 9: The tertiary structure model of the Muc1 protein molecule and the diagram of the finally selected antigen peptide.

[0037] Figure 10 Western blot analysis of the binding of Muc1-specific antibody to native tissue proteins of turbot intestine, where M represents the marker and NC represents the negative control.

[0038] Figure 11 : Indirect immunofluorescence assay of the specific binding reaction of Muc1 antibody with mucin Muc1 in the gills, hindgut, skin and olfactory sac of turbot (40× objective). Neg (Negetive) indicates a negative control with rabbit negative serum instead of Muc1 antibody (mucus cells stained with WGA). Detailed Implementation

[0039] This invention utilizes bioinformatics techniques to analyze the antigenic characteristics of the Muc1 molecule, screens for dominant antigenic epitopes, and prepares a specific antibody with high recognition activity. This antibody can effectively label Muc1-positive mucus cells in turbot mucosal tissue and Muc1 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.

[0040] The prepared antibody specifically binds to the Muc1 antigenic peptide. Western blot results showed that the antibody specifically recognizes the native Muc1 protein in the intestinal tissue of turbot, while no binding signal was observed in the rabbit negative serum control, indicating good reaction specificity. Immunofluorescence combined with WGA staining further confirmed that the antibody can accurately label the cell membranes of WGA-positive mucin cells in the gills, hindgut, skin, and olfactory sac tissues of turbot, achieving in situ localization of the target cells.

[0041] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0042] Example 1: Gene cloning of the Muc1 molecule in turbot

[0043] (1) Primer design for conserved gene sequences

[0044] Based on sequence information from the turbot genome database, specific primers were designed and synthesized for PCR amplification using cDNA from the hindgut tissue of turbot as a template. During the amplification of the turbot Muc1 gene sequence, it was found that the posterior region of Muc1 is a highly repetitive tandem repeat domain. Due to the presence of this structure, the PCR amplification products exhibited significant banding, making it impossible to obtain a single target band. After cloning the amplified products, despite multiple screenings, the obtained positive clones, upon sequencing verification, all showed varying degrees of sequence truncation or deletion, failing to yield the target fragment containing the complete posterior sequence. Ultimately, this study only successfully obtained the coding sequence of the front part of the gene. The amplified products were purified and ligated into the pMD19-T vector, transformed into DH5α competent cells, and positive clones were screened by colony PCR and sequenced. The sequencing results showed that the obtained cDNA fragment was 1057 bp in length, and BLAST alignment completely matched the predicted partial CDS sequence of the turbot Muc1 gene, confirming that this fragment belongs to the coding region of the turbot Muc1 gene.

[0045] Gene structure analysis revealed that the Muc1 gene in turbot contains three exons and two introns. Domain prediction showed that its encoded product possesses a phospholipase C catalytic X domain (PLCXc) and an RPT1 internal repeat domain. SignalP and TMHMM analysis indicated that amino acids 1-19 at the N-terminus of this protein form a signal peptide sequence and that a transmembrane region is present, consistent with typical characteristics of membrane-bound mucins. A phylogenetic tree constructed using the neighbor-joining method showed that Muc1 in bony fishes clustered into independent branches, with the clustering pattern largely consistent with species evolutionary relationships. The turbot Muc1 is most closely related to the brown-spotted grouper (Epinephelus fuscoguttatus). Indirect ELISA analysis confirmed this relationship.

[0046] Based on the CDS sequence of the Muc1 gene in turbot published in the NCBI database, specific amplification primers were designed using Primer Premier 5.0 software. During primer design, key parameters such as secondary structure formation potential (e.g., dimers, hairpin structures) and annealing temperature were comprehensively evaluated and optimized. The final primer sequences were determined as follows: forward primer: 5'-ACAGGTTTCAATGATGACCCAG-3'; reverse primer: 5'-CTCTAGCTTGGGCATCGCTTC-3', synthesized by Qingke Biotechnology Co., Ltd.

[0047] (2) Amplification of the core gene fragment

[0048] cDNA synthesized from total RNA reverse transcribed from turbot tissue was used as a template for PCR amplification using specific primers designed for the front segment of the Muc1 gene CDS region. After the reaction, the amplified product was separated by 1.0% agarose gel electrophoresis. The target band with the expected molecular weight was observed and recovered under a gel imaging system, purified by gel recovery, and ligated into the pMD19-T vector, which was then transformed into E. coli DH5α competent cells. Positive clones were screened by colony PCR, and single colonies were sent to Qingke Biotechnology Co., Ltd. for bidirectional sequencing. The obtained sequences were compared with the NCBI database by BLAST. The results showed that the amplified product was 1057 bp in length, consistent with the published Muc1 front segment sequence, with no mismatches or gaps.

[0049] (3) Bioinformatics analysis of the Muc1 protein sequence of turbot

[0050] BLAST results showed that the Muc1 protein possesses the typical PLCXc and RPT1 domains of membrane-bound mucins. A phylogenetic tree of the turbot Muc1 molecule was constructed using MEGA 5.0 software. Figure 1 Multiple sequence alignment analysis of the obtained turbot Muc1 protein sequence with sequences from other species was performed using DNAMAN software. The results showed that the turbot Muc1 molecule had the highest sequence homology with the Muc1 molecule of the brown-spotted grouper (Epinephelus fuscoguttatus).

[0051] Example 2: Screening of Muc1 antigenic peptide epitopes in turbot

[0052] (1) The transmembrane structure of the turbot Muc1 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 a transmembrane domain, which conforms to 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 Muc1 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 ).

[0053] (2) To analyze the structural features of the Muc1 protein, its amino acid sequence was functionally identified using an online prediction tool. The results showed that ( Figure 4This protein contains several characteristic structural units: a typical PLCXc domain (positions 49-184) and two RPT1 domains (positions 331-781 and 779-1313).

[0054] (3) The number of exons and introns in the Muc1 gene of turbot are 3 and 2, respectively. Figure 5 ).

[0055] (4) Analysis of the secondary structure of the turbot Muc1 protein showed that the β-sheet and random coil sections exhibited good immunogenicity and reactivity; α-helices accounted for 5.47%, extended chains for 2.11%, and random coils for 92.42%. The distribution of various structures in the amino acid sequence of turbot Muc1 is shown in [Figure number missing]. Figure 6 .

[0056] (5) Analysis of the antigens of the Muc1 protein revealed that the B-cell linear antigenic site sequence of the Muc1 protein is mainly located at amino acid residues such as 18-31 and 388-1347. Figure 7 ).

[0057] (6) The amino acid sequence of the turbot Muc1 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, and 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 Muc1 molecule. Figure 8 ).

[0058] A three-dimensional structural model of the turbot Muc1 protein was constructed using the AlphaFold3 deep learning system. The predicted model was finely optimized and rendered using PyMOL software, and compared with known crystal structures to clarify the unique structural domains of the target protein. Subsequently, previously screened antigenic epitopes were mapped onto this 3D model and highlighted as chromospheres. Structural localization analysis showed that the candidate epitopes are exposed on the protein molecule surface, possess high solvent accessibility, and meet the basic structural requirements for antibody recognition targets. Figure 9 ).

[0059] Example 3: Preparation of Muc1-specific antibody against turbot

[0060] (1) Determination of antigenic peptide sites

[0061] The screening process for dominant antigenic epitopes is as follows: Based on the predicted data from Example 2, non-extracellular fragments located in signal peptides, transmembrane regions, and intracellular regions are first eliminated; combining antigen parameters and secondary structure (random coil, β-turn) analysis, candidate B-cell linear epitopes are identified. Spatial localization verification is performed using a three-dimensional model constructed with AlphaFold3, and the final sequence is determined. 64 TTEPKPKPTSSTTEQ 78 It is a targeted epitope because it is located on the protein surface and has an open spatial conformation. After chemical synthesis and conjugation with a hemocyanin carrier, mass spectrometry analysis showed that its purity met the standards for immunogen preparation.

[0062] (2) Immunity

[0063] New Zealand white rabbits were immunized with a Muc1 antigen peptide-hemocyanin (KLH) conjugate complex to prepare specific polyclonal antibodies. The immunization program employed a strategy of primary immunization followed by four booster immunizations, totaling five immunizations with a 7-day interval between each. 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 sites on the back and groin, with 100 μL injected at each site. Three days after the final 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 following day, the blood was centrifuged at 8000 g for 15 minutes at 4°C, and the supernatant was carefully aspirated, aliquoted, and stored at -80°C to obtain rabbit anti-Muc1 specific antibodies.

[0064] Example 4: ELISA determination of Muc1 antibody titer

[0065] (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 Muc1 antigen peptide.

[0066] (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.

[0067] (3) Repeat the washing process above, add 100 μL Muc1 antibody to each well, and incubate at 37 °C for 1 h.

[0068] (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.

[0069] (5) Add 100 μL of pNPP chromogenic solution, incubate in the dark for 10 min, preheat the microplate reader for 30 min, and then detect OD 405. Repeat 20 cycles. The result showed that the Muc1 antibody titer was 1:16000.

[0070] Example 5: Identification of Muc1-specific antibodies by Western blot

[0071] (1) SDS-AGE electrophoresis

[0072] 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.

[0073] 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.

[0074] 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.

[0075] ④ Place the assembled transfer clamp into the electrotransfer cell, 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.

[0076] ⑤ After the transfer is complete, remove the PVDF membrane.

[0077] (2) Immunoblotting:

[0078] ① Wash the PVDF membrane with PBS for 15 min, then block it in 5% BSA for 1 h at 37 ℃;

[0079] ② Wash three times with PBST, 5 min each time; add turbot Muc1 antibody and incubate at 37 ℃ for 1 h. Use rabbit negative serum as a negative control;

[0080] ③ Wash three times using the same method as ②;

[0081] 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.

[0082] ⑤ Wash three times using the same method as in ②;

[0083] 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.

[0084] The recognition ability of the prepared rabbit anti-turbot Muc1 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 140 kDa, while the negative control showed no color signal at this position. Figure 10 This indicates that the prepared antibody can specifically recognize the natural Muc1 protein in turbot intestine tissue.

[0085] Example 6: Indirect immunofluorescence identification of turbot Muc1 antibody

[0086] ① Healthy turbot (body weight 60±5 g) were selected as the research subjects and their tissues were collected after one week of temporary rearing. After collecting gill, hindgut, skin, and olfactory sac tissues, they were immediately rinsed in 0.01 M phosphate-buffered saline (PBS, pH 7.4) to remove impurities. Excess moisture on the tissue surface was then absorbed with qualitative filter paper, and the tissue blocks were placed in a square foil box pre-filled with OCT embedding medium. The orientation was precisely adjusted using a dissecting needle to ensure proper embedding. Finally, the embedding box was rapidly transferred to a -80 ℃ freezer for quick-freezing and storage for subsequent tissue section preparation.

[0087] ② Fix the embedded tissue block onto the sample holder of the cryostat, set the section thickness to 6 μm, and cut 4 tissue sections consecutively. Gently attach the sections to the cryostat using an adhesive slide. Immediately immerse the sections in pre-cooled acetone at 4 ℃ for 15 min. After fixation, remove the sections and place them in a fume hood to dry for 15 min. After the acetone has completely evaporated, transfer them to a -20 ℃ freezer and seal for later use.

[0088] ③ Remove the frozen sections from the -20℃ freezer and allow them to thaw at room temperature for 20 min. Transfer the sections to a staining jar containing PBST and rinse three times on a shaker for 5 min each time. Remove the sections, gently shake off any residual liquid, and wipe the tissue periphery dry with absorbent paper. Immediately use an immunohistochemical pen to delineate a hydrophobic barrier around the tissue periphery. Add sufficient 5% BSA blocking solution (solvent: 0.01 M PBS) to the area within the barrier, then transfer the sections to a humidified chamber and incubate at 37℃ for 1 h.

[0089] ④ After completing the blocking step, discard the blocking solution and spin-dry the sections. Dilute rabbit anti-turbot Muc1 serum at a ratio of 1:1000 with PBST buffer (PBS containing 0.05% Tween-20) to prepare the primary antibody working solution. Add this working solution evenly to the tissue area, ensuring complete coverage of the section surface. Place the sections in a humidified chamber and incubate at 37°C for 1 h. After incubation, discard the primary antibody solution, then place the sections on a shaker and wash thoroughly three times with PBST buffer for 5 min each time to remove unbound antibody residue and nonspecific background.

[0090] ⑤ After incubation with the primary antibody and washing, place the slides in a centrifuge to remove residual liquid from the slide surface. Then, 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, used for staining mucin) diluted 1:1000 with PBS. 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.

[0091] 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 it as above.

[0092] ⑦ After drying, add anti-fluorescence quenching mounting medium in the dark, and then cover with a coverslip.

[0093] ⑧ Results observed under a fluorescence microscope.

[0094] To evaluate the tissue recognition efficacy of the prepared rabbit anti-turbot Muc1 antibody, indirect immunofluorescence was used for detection. The results showed that the antibody specifically targets endogenous Muc1 molecules in the gills, hindgut, skin, and olfactory sac tissues, eliciting bright positive fluorescent signals. Figure 11The signal-enriched regions were primarily found on the cell membranes of mucin cells, exhibiting ring-like or sheet-like aggregations. Fluorescence was also observed on the free surfaces of some epithelial cells. Further verification of the antibody's recognition specificity was achieved through co-localization analysis of immunofluorescence and WGA: WGA, as a specific probe for mucin glycosylation sites, showed a high degree of co-localization with the Muc1 antibody fluorescence in the mucin cell membrane region. In the control experiment, tissue sections treated with FITC-WGA-counted rabbit negative serum showed only WGA-positive fluorescence and no Muc1-positive fluorescence, ruling out interference from non-specific binding. These results strongly confirm the antibody's precise recognition ability of the membrane-bound mucin Muc1.

[0095] In summary, this invention provides a specific antibody against Muc1, a membrane-bound mucin found in turbot. This antibody can serve as a molecular recognition probe to detect changes in Muc1 expression in turbot mucosal tissue under pathogen infection or vaccine-induced immunization conditions, thereby enabling the analysis and evaluation of mucosal barrier function. The acquisition of this antibody provides an important experimental tool for studying fish mucosal immune mechanisms, a technical means for evaluating the efficacy of mucosal vaccines, and a technological foundation for research on fish disease control strategies based on mucus barrier function regulation. This is of great significance for promoting the healthy and sustainable development of the turbot aquaculture industry.

Claims

1. An antigenic epitope polypeptide, characterized in that, The amino acid sequence of the antigenic epitope polypeptide is SEQ ID NO:

1.

2. A coupling protein, characterized in that, The conjugated protein is obtained by conjugating the C-terminus of the antigenic epitope polypeptide of claim 1 with a KLH carrier protein.

3. A polyclonal antibody, characterized in that, The polyclonal antibody is prepared by conjugating an antigenic epitope polypeptide with the amino acid sequence SEQ ID NO:1 to a carrier protein, immunizing New Zealand white rabbits, and then collecting serum; the carrier protein is a KLH carrier protein.

4. The use of the polyclonal antibody according to claim 3 in the preparation of a reagent for identifying Muc1-positive mucinous cells of turbot.

5. A kit for detecting or tracing Muc1-positive mucinous cells in turbot, characterized in that, The kit contains the polyclonal antibody as described in claim 3.

6. The reagent kit as described in claim 5, characterized in that, The kit described is a paraffin or frozen section detection kit.

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