Preparation and application of specific antibody against Muc5AC of flounder

Abstract

The present invention provides a specific antibody of the oval flounder mucin Muc5AC and a preparation and application method thereof. The oval flounder Muc5AC specific antibody provided can be used to detect oval flounder Muc5AC and its positive mucus cells, providing a key tool for studying the characteristics of oval flounder mucus cells and mucins and its mucosal immune defense mechanism. The present invention screened and obtained the B cell antigen epitope STTEKVKTTESPKST of the oval flounder mucin Muc5AC gene, coupled with KLH and then immunized Balb / c mice, thereby preparing the oval flounder Muc5AC specific antibody, providing an important tool for detecting oval flounder Muc5AC protein molecules and its positive mucus cells.

Description

Technical Field

[0001] The invention belongs to the technical field of fish immunology, and particularly relates to a specific antibody of Paralichthys olivaceus mucin Muc5AC and a preparation method and application thereof. Background Art

[0002] The epithelial tissue, mucus and symbiotic microorganisms on the surface of fish mucosa constitute a mucosal barrier that blocks pathogen infection and is the first line of defense for fish against pathogen invasion. The mucus layer covers the fish epithelium and is in direct contact with the outside world, forming a dynamic protective structure, which can produce an active immune response to changes in the external environment, physical interference and immune stimulation. Fish mucus contains a wide range of natural immune factors, which play a unique physical and chemical barrier function. Among them, mucin secreted by mucus cells is the most important component of mucus, which is mainly divided into gel-forming mucin and transmembrane mucin. Gel-forming mucin represented by Muc5AC is a key molecule to maintain mucosal homeostasis, which can fix immune active molecules and participate in inflammation, repair or healing reactions. Muc5AC contains sugar chains of antigen ligands, which can serve as an ideal binding site for many symbiotic bacteria, inhibit infection and eliminate pathogens through competition between beneficial bacteria and pathogens. Therefore, the analysis of the gene structure of mucin Muc5AC is helpful for its functional research and the design and development of artificially synthesized mucin drugs / vaccines. However, the long nucleic acid sequence of mucin genes and the presence of multiple tandem repeat sequences make gene cloning one of the difficulties in mucin research. At present, only a few teleost mucin genes have been described, and they are partial cDNA fragments. In the NCBI database, most mucins only provide predicted sequences. In addition, due to the lack of fish mucin-specific labeling probes, fish mucin research is limited to mucus polymer sugar chains, and research on mucus cells is limited to morphological studies using traditional staining methods such as Alcian blue staining (AB staining) and periodic acid Schiff staining (AB-PAS staining). There is a lack of information on the cloning and expression analysis of the fish mucin Muc5AC gene, and functional research at the protein level is still blank.

[0003] Studies in mammals have found that excessive or ectopic expression of mucins is associated with many diseases and is often used for disease diagnosis or differential diagnosis. Recombinantly expressed mucin fragments can be used as vaccines for treatment, such as small mucin peptides used in anti-tumor vaccines, and can also be used as immunomodulators. Studies in fish have found that mucus secretion increases after pathogen infection, but research on the application of mucins in fish disease prevention and control is still scarce. Therefore, it is crucial to recombinantly express mucin genes, analyze the structural characteristics of mucins, and develop efficient mucin-specific antibodies. Paralichthys olivaceus is an important economic fish in my country, but diseases have caused significant economic losses to the paralichthys industry. Cloning and expressing the Muc5AC gene of Paralichthys olivaceus, obtaining recombinant expressed proteins, and preparing specific antibodies will help study the dynamic changes of mucins and mucus cells under the stimulation of different pathogens and immunogens at the gene and protein levels, explore the application potential of mucin Muc5AC in disease prevention and control and immune defense, and provide strong support for in-depth understanding of the fish mucosal immune defense mechanism and mucosal vaccine development. Summary of the invention

[0004] The purpose of the present invention is to provide a specific antibody for the otter mucin Muc5AC and a preparation method and application thereof. The provided specific antibody can be used to mark the otter mucin Muc5AC and its positive mucus cells, provide a key probe for studying the characteristics of otter mucus cells and mucins, and provide an efficient immunological reagent for analyzing the otter mucosal immune defense mechanism.

[0005] The invention provides a B cell antigen epitope polypeptide for preparing a Japanese flounder Muc5AC-specific antibody, wherein the specific amino acid sequence of the polypeptide is STTEKVKTTESPKST (SEQ ID NO: 1).

[0006] The present invention also protects the use of the provided antigen epitope polypeptide in the preparation of fish mucin vaccines or immunomodulators;

[0007] The present invention also protects a derivative polypeptide of the B-cell antigen epitope polypeptide having the amino acid sequence of SEQ ID NO: 1, that is, a polypeptide obtained by replacing, deleting or adding one or more amino acids;

[0008] The present invention also protects a coupling protein, which is a functional molecule for enhancing immune effect coupled to the 5′ end or 3′ end of the B cell antigen epitope polypeptide with the amino acid sequence of SEQ ID NO: 1;

[0009] As a specific record of the embodiment, the functional molecule is a KLH (hemocyanin) carrier;

[0010] The present invention also provides a specific antibody, which is prepared by immunizing an animal with the above-mentioned antigen polypeptide;

[0011] The animals described, as specifically described in the examples, are Balb / c mice;

[0012] The method for preparing the specific antibody of the present invention comprises the following steps: coupling the B cell antigen epitope of flounder Muc5AC with a carrier, immunizing Balb / c mice, and collecting serum for post-processing to obtain the anti-flounder Muc5AC specific antibody.

[0013] The carrier, as specifically described in one embodiment, is a KLH (hemocyanin) carrier;

[0014] The invention discloses an application of the specific antibody of the flounder Muc5AC molecule in the preparation of an identification reagent for the flounder mucin Muc5AC.

[0015] The invention discloses an application of the specific antibody against the flounder Muc5AC molecule in preparing a reagent for studying the immune response of Muc5AC-positive mucous cells.

[0016] The present invention also provides a kit for detecting or tracing Muc5AC and its positive mucus cells of Japanese flounder in paraffin or frozen sections, characterized in that the kit comprises:

[0017] 1) Antigen retrieval solution (0.01 M sodium citrate buffer, pH 6.0) is used to open the cross-linking bonds formed between aldehydes and antigenic determinants under high temperature to expose the antigenic epitopes;

[0018] 2) 5% bovine serum albumin (BSA) blocking solution (prepared in 0.01 M PBS), used to block nonspecific binding sites to reduce nonspecific adsorption;

[0019] 3) Antibody specific to the Muc5AC molecule of Japanese flounder (1:500, diluted in 0.01M PBS), used to identify Muc5AC-positive mucous cells in the mucosal tissue of Japanese flounder;

[0020] 4) Cy3-labeled goat anti-mouse IgG antibody (1:1000, diluted in 0.01 M PBS) was used to specifically recognize Muc5AC-specific antibodies and visualize Muc5AC-positive mucus cells;

[0021] 5) DAPI (1:2000, diluted in 0.01 M PBS), for cell nucleus staining.

[0022] The components of the 0.01M sodium citrate buffer solution (pH 6.0): 3.8mL of 0.1M citric acid solution and 16.2mL of 0.1M sodium citrate buffer solution were mixed and then distilled water was added to 200mL;

[0023] The 0.01M PBS (1 L, pH 7.4) contained the following ingredients: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and HCl was used to adjust the pH to 7.4.

[0024] The present invention cloned the flatfish mucin Muc5AC gene, screened and obtained the B cell antigen epitope of the flatfish mucin Muc5AC gene, and used the antigen epitope sequence to prepare antigens and specific antibodies to the flatfish mucin Muc5AC, which provides the possibility of detecting the expression changes of the flatfish mucin Muc5AC at the gene and protein levels, provides a key tool for detecting the flatfish Muc5AC positive mucus cells, and provides basic data for the research and development of mucin vaccines / drugs, etc. The present invention realizes the identification of Muc5AC positive mucus cells and subsequent functional research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 :MEGA5.0 software was used to construct a phylogenetic tree of Muc5AC molecules using the neighbor-joining method;

[0026] Figure 2 : Analysis of transmembrane sequence and signal peptide sequence of flounder Muc5AC;

[0027] Figure 3 :Analysis of protein domain structure of flounder Muc5AC;

[0028] Figure 4 :Sequence map of the PTS domain of the Japanese flounder Muc5AC molecule.

[0029] Figure 5 :The predicted B cell linear antigen epitope of flounder Muc5AC predicted and analyzed by IEDB software;

[0030] Figure 6 :The secondary structure domain diagram of flounder Muc5AC;

[0031] Figure 7 :Analysis of antigen epitope parameters of flounder Muc5AC molecule predicted by DNAStar software;

[0032] Figure 8 :Model diagram of the tertiary structure of Muc5AC protein molecule;

[0033] Fig. 9:Western blot analysis of the binding of Muc5AC-specific antibody to natural mucin, where M represents marker and NC represents negative control;

[0034] Fig.10 : Indirect immunofluorescence analysis of the specific antibody and the gill and skin mucous cells of the Japanese flounder (objective magnification 40×), NC represents the negative control. DETAILED DESCRIPTION

[0035] The flounder mucin Muc5AC gene screened and obtained by the present invention can be used as a marker of mucus cells and can be used to identify Muc5AC positive mucus cells. The flounder Muc5AC specific antibody of the present invention can be used as a key tool for in-depth research on the functional characteristics of flounder mucus cells, thereby evaluating the immune response level of fish mucus cells.

[0036] The objective of the present invention is achieved through the following technical solutions:

[0037] The gene sequence and amino acid sequence structure of the flounder Muc5AC molecule were obtained by gene cloning and verified by bioinformatics analysis. After antigenicity analysis, amino acid sequences with high antigenicity were screened for peptide synthesis.

[0038] The results of enzyme-linked immunosorbent assay showed that the specific antibody of Muc5AC molecule of Japanese flounder could specifically bind to the corresponding synthetic peptide.

[0039] The results of protein immunoblotting experiments showed that the specific antibodies against Muc5AC molecule of Japanese flounder could specifically bind to natural mucins in skin and gill mucus.

[0040] The results of indirect immunofluorescence experiments showed that the specific antibodies of the flounder Muc5AC molecule specifically bound to the mucin Muc5AC in the gills and skin of the flounder, showing a positive fluorescence signal. Under a fluorescence microscope, it can be observed that the fluorescence signal is distributed in clusters in the mucus cells and in scattered spots in the interstitial space.

[0041] The gene cloning method of the Japanese flounder Muc5AC molecule of the present invention is to use bioinformatics software to analyze the conservative region of the published fish mucin Muc5AC protein sequence, select two peptide segments of 6 consecutive identical amino acids in the protein comparison sequence, and the distance between the peptide segments is greater than 20 amino acids. Degenerate primers are designed for them, and the primers are optimized in combination with the preference of Japanese flounder codons. The core sequence of Muc5AC is obtained by PCR amplification using the gills, skin and intestines of Japanese flounder as templates, and a partial cDNA sequence of the Muc5AC gene with a size of 4788bp is obtained by PCR amplification by designing specific primers (GSP) and using RACE technology.

[0042] The cloned Muc5AC gene of the present invention was compared by bioinformatics, and the results showed that it was the Japanese flounder Muc5AC gene, which had typical mucin vWD (von Willebrand Factor), C8 (conserved 8cysteines domain) and TIL domain (Trypsin Inhibitor like cysteine ​​rich domain). In addition, Muc5AC has a sequence rich in PTS amino acids (proline, threonine, serine) that is unique to mucin. SignalP and TMHMM analysis results showed that Muc5AC had a signal peptide of about 24 amino acids at the N-terminus, and did not have a transmembrane region and an intracellular region, proving that Muc5AC is a secretory protein. The phylogenetic tree of Japanese flounder Muc5AC was analyzed using the NJ method, and the results showed that Japanese flounder Muc5AC could be clustered into one branch with Muc5AC of bony fish, and its similarity with turbot (Scophthalmus maximus) was the highest.

[0043] The results of indirect enzyme-linked immunosorbent assay showed that the Muc5AC-specific antibody of Japanese flounder could specifically bind to the naked peptide of Muc5AC of Japanese flounder. The results of immunoblotting showed that the Muc5AC-specific antibody of Japanese flounder could recognize the natural mucin of gills and skin, while the negative serum of mice as a control could not bind to the natural mucin. The results of indirect immunofluorescence assay showed that the Muc5AC-specific antibody of Japanese flounder could specifically recognize the cytoplasmic contents of Muc5AC-positive mucus cells in gills and skin tissues and Muc5AC in the interstitial space.

[0044] The present invention is described in detail below in conjunction with embodiments and drawings.

[0045] Example 1: Gene cloning of the Japanese flounder Muc5AC molecule

[0046] (1) Designing degenerate primers for the conserved sequence of the gene

[0047] The Muc5AC protein sequences of other fish species published on NCBI were searched, and sequences of fish species with high homology to the Japanese flounder were searched through Blast. The more conservative regions in the sequences (more than 6 consecutive identical amino acids) were searched using BioEdit. Degenerate primers were designed based on the conserved regions, and the degenerate primers were optimized based on the codon preference of the Japanese flounder and sent to Qingke Company for synthesis.

[0048] (2) Gene core fragment amplification

[0049] Degenerate primers were used to amplify the Muc5AC gene using flounder cDNA as a template. The PCR process was optimized by combining temperature gradient PCR and Touchdown PCR methods until the core fragment was amplified. The amplified PCR product was detected by agarose gel electrophoresis, and the gel imaging results were observed under ultraviolet light. The gel where the target band was located at the estimated size was cut and recovered, connected to the T vector, transferred into cloning bacteria, and single clone colonies were picked and sent to the company for sequencing. The sequence results returned by sequencing were verified by Blast on NCBI.

[0050] (3) RACE to obtain partial cDNA sequence of flounder Muc5AC

[0051] The core sequence was amplified by 3′-RACE and 5′-RACE to obtain the full length of Muc5AC gene. Specific primers were designed (Table 1) to amplify the gene sequence of flounder Muc5AC with a size of 4788 bp.

[0052] Table 1: Muc5AC gene RACE specific primers

[0053]

[0054] BLAST results showed that the Muc5AC protein had typical mucin vWD, C8 and TIL domains, and also had a conserved domain rich in PTS amino acids that is unique to mucins. The amino acid sequence of the PTS domain of the Japanese flounder Muc5AC molecule was obtained. Using MEGA5.0 software, a phylogenetic tree of the Japanese flounder Muc5AC molecule was constructed ( Figure 1 ); DNAMAN software was used to perform multiple sequence alignment analysis on the obtained protein sequence of the Japanese flounder Muc5AC molecule and the sequences of other species. The results showed that the sequence homology between the Japanese flounder Muc5AC molecule and the turbot Muc5AC was the highest.

[0055] Example 2: Screening of the epitope of the oligoflounder Muc5AC antigenic peptide site

[0056] (1) The TMHMM website analysis confirmed that the Muc5AC protein had no transmembrane domain and intracellular region and was a secretory protein ( Figure 2 Signal IP (https: / / services.healthtech.dtu.dk / service.php?SignalP-5.0) was used to analyze the signal peptide sequence of Muc5AC protein. The analysis results showed that the 1st to 24th amino acids at the end of Muc5AC protein were signal peptide sequences ( Figure 2 ).

[0057] (2) Analysis of the Muc5AC protein domain, Figure 3The results showed that the 39-193 and 855-1013 amino acids of Muc5AC protein folded into a typical mucin vWD domain (von Willebrand factor (vWF) type D domain), the 224-299, 588-662 and 1049-1123 amino acids folded into a C8 domain (conserved 8cysteines domain), the 360-402 and 727-790 amino acids folded into a mucin vWC domain (von Willebrand factor (vWF) type Cdomain), and the TIL domain (Trypsin Inhibitor like cysteine ​​rich domain) and the PTS (proline, threonine, serine)-rich amino acid sequence unique to mucins were also found. Figure 4 ).

[0058] (3) Analysis of the antigenic sites of Muc5AC protein revealed that the B cell linear antigenic site sequences of Muc5AC protein are mainly 183-196, 204-228, 313-331, 381-406, 808-827, 999-1016, 1035-1052, 1291-1343 ( Figure 5 ).

[0059] (4) The secondary structure of the flounder Muc5AC protein was analyzed. The results showed that the β-fold and random coil of the secondary structure had good immunogenicity and reactivity; α-helix accounted for 11.78%, extended chain accounted for 24.25%, β-turn accounted for 6.33%, and random coil accounted for 57.64%. The distribution of various structures in the amino acid sequence of flounder Muc5AC is shown in Figure 6 .

[0060] (5) DNAStar software was used to analyze the sequence of Muc5AC of Japanese flounder. The analysis parameters mainly included hydrophilicity (Hydrophilicity Plot-Kyte-Doolittle), flexibility (Flexible Regions-Karplus-Schulz), antigenicity (Antigenic Index-Jameson-Wolf), and surface accessibility (Surface Probability Plot-Emini). Based on the above analysis, the amino acid segments with hydrophilicity index ≥ 0, surface accessibility index ≥ 1, and antigenicity index ≥ 0 in the β-turn and random coil regions were selected as potential epitopes. Figure 7 The suitable sites in the antigenic epitope of Muc5AC protein molecule of Japanese flounder can be found.

[0061] (6)Use SWSS( SWISS-MODEL Interactive Workspace(expasy.org) ) Analysis of the three-dimensional structure of the flounder Muc5AC protein ( Figure 8 ).

[0062] Example 3: Preparation of Specific Antibodies to the Japanese Flounder Muc5AC Molecule

[0063] (1) Determine the antigenic peptide site

[0064] The predicted data in Example 2 were combined to finally determine the site of the antigenic peptide. First, the antigenic epitopes located in the signal peptide, transmembrane region and intracellular region were excluded; secondly, the antigenic epitope parameters and the predicted potential B cell antigen linear epitopes were analyzed; finally, the antigenic epitopes located in the random coil and β-turn regions in the secondary structure were analyzed to determine the specific location of the antigenic peptide. The three-dimensional structure of the flounder Muc5AC protein molecule was constructed, and the flounder Muc5AC antigenic peptide was finally selected. 1297 STTEKVKTTESPKST 1311 Located on the surface of protein molecules, it can be used to prepare Muc5AC-specific antibodies. The antigen peptide is synthesized by the company and coupled to hemocyanin, and its purity is detected by mass spectrometry.

[0065] (2) Immunity

[0066] Balb / c mice were immunized with the above-mentioned flounder Muc5AC antigen peptide-KLH coupled carrier complex as an antigen. Each immunization dose was 100 μL, and the immunization was performed 4 times, wherein the basic immunization and booster immunization were intraperitoneal injections with an interval of 14 days; the second booster immunization and the third booster immunization were tail vein injections with an interval of 7 days.

[0067] ① Basic immunization: Muc5AC antigen peptide-KLH complex and Freund's complete adjuvant were mixed in equal amounts (V / V) as antigen;

[0068] ②Boosting immunization: Muc5AC antigen peptide-KLH complex and Freund's incomplete adjuvant were mixed in equal amounts (V / V) as antigen;

[0069] ③ Second booster immunization: Muc5AC antigen peptide-KLH complex adjuvant-free protein suspension;

[0070] ④ Second boost immunization: Muc5AC antigen peptide-KLH complex adjuvant-free protein suspension;

[0071] ⑤ After the last immunization, blood was collected from the eyeballs 3 days later. The collected blood was tilted and left to stand for 2 hours at room temperature, and then placed in a 4°C refrigerator overnight. The next day, the centrifuge tube was centrifuged at 4°C and 8000g for 10 minutes, and the supernatant was carefully aspirated to obtain specific antibodies.

[0072] Example 3: ELISA determination of the titer of Muc5AC antibody

[0073] ① Adjust the antigen concentration to 50 μg / mL with PBS and add 100 μL to each well of a 96-well plate, with 3 replicates. The negative control uses mouse negative serum, and the positive control is coated with an irrelevant protein and incubated with the Muc5AC antigen peptide-KLH complex.

[0074] ② Coat overnight at 4℃, pour off the coating solution the next day, gently add 100μL PBST to the surface, shake and wash on a decolorizing shaker, repeat 3 times, 5 minutes each time. Tap off the liquid on the ELISA plate, add 100μL BSA solution, and block at 37℃ for 2h.

[0075] ③ Repeat the last washing process, add 100 μL Muc5AC antibody to each well, and incubate at 37°C for 1 hour.

[0076] ④ Wash again with PBST for 3 times, spin dry, add 100 μL AP secondary antibody, incubate at 37°C for 1 hour, and wash again.

[0077] ⑤ Add 100 μL pNPP color development solution, color for 10 min in the dark, preheat the microplate reader for 30 min and detect OD 405, for a total of 20 cycles.

[0078] Results: The titer of Muc5AC antibody was measured to be 8000.

[0079] Example 4: Identification of Flounder Muc5AC-specific Antibodies by Immunoblotting

[0080] (1) SDS-UAgPAGE electrophoresis

[0081] ① The natural gill mucin and skin mucin of Japanese flounder were added to sample buffer containing sodium dodecyl sulfate (SDS) in equal proportions, reduced at 95°C for 30 min, and iodoacetamide was added to a final concentration of 25 mM, and incubated at room temperature in the dark for 1 h;

[0082] ② Add the sample treated in ① to the sample loading wells, add 10μL of sample to each well. Use low current (30-40mA) at the beginning, and increase the current (50-70mA) after the sample is aggregated into a line in the concentrated gel. Stop the electrophoresis when the bromophenol blue indicator reaches the bottom edge of the gel plate, remove the gel, and place it in Coomassie Brilliant Blue dye solution for staining for 1h.

[0083] ③ Cut a piece of nitrocellulose membrane (pore size 0.22μm) of the same size as the gel, wet it with electrotransfer buffer (electrotransfer buffer: 25mmol / L Tris-Base, 192mmol / L glycine, 20% methanol, pH 8.3), and place it on the gel. Cut off a corner of the nitrocellulose membrane to mark the start of the sample sequence. Support it with moistened filter paper and stick the second moistened filter paper on the other side; so that the gel block, nitrocellulose membrane and filter paper form a sandwich.

[0084] ④ Place the "gel sandwich" assembly into an electrophoresis tank filled with electrotransfer buffer, with the nitrocellulose membrane facing the anode; select 200 mA for electrophoresis constant current and perform electrophoresis for 5 hours;

[0085] ⑤After the transfer is completed, take out the nitrocellulose membrane.

[0086] (2) Immunoblotting

[0087] ① Wash the nitrocellulose membrane with PBS for 10 min, then block it in 3% BSA for 1 h at 37°C;

[0088] ② Wash with PBST 3 times, 5 min each time; add flounder Muc5AC antibody and incubate at 37℃ for 1 h. Mouse negative serum was used as negative control;

[0089] ③ Wash 3 times in the same way as ②;

[0090] ④ Add alkaline phosphatase-labeled goat anti-mouse Ig antibody (1:4000 dilution) to the nitrocellulose membrane and shake slowly at 37°C for 1 hour;

[0091] ⑤ Wash 3 times in the same way as ②;

[0092] ⑥Put the nitrocellulose membrane into alkaline phosphatase colorimetric solution (NBT-BCIP colorimetric solution) until the color is clear;

[0093] ⑦ Wash with deionized water to terminate the reaction. Sandwich the nitrocellulose membrane between filter papers, let it stand to dry, and store it in a dark place.

[0094] ⑧ Meanwhile, boil the gel in Coomassie Brilliant Blue in boiling water until the bands are clear. Take photos and record.

[0095] Results: The prepared Muc5AC antibody specifically binds to the natural mucin in gills and skin of Japanese flounder. The electrophoretic bands show a tailing effect unique to mucin. This is due to the difference in mucin glycosylation and the fact that mucin, as a high molecular weight glycoprotein, is blocked when passing through the gel pores during electrophoresis. Fig. 9 ).

[0096] Example 5: Identification of the Japanese flounder Muc5AC antibody by indirect immunofluorescence method

[0097] ①Take the frozen sections of gills and skin tissues of Japanese flounder out of the -20℃ refrigerator and rewarm them for 10 minutes. Wash them with PBST for 3 times, 5 minutes each time. After drying, circle them with an immunohistochemical pen, add 5% BSA, and incubate them in a wet box at 37℃ for 1 hour.

[0098] ② After drying BSA, add mouse anti-flounder Muc5AC serum diluted 1:200 as the primary antibody, and mouse negative serum as the negative control. Incubate in a wet box at 37℃ for 1.5h. Wash with PBST for 3 times, 5min each time, to fully wash away the unbound primary antibody.

[0099] ③ Use a slide spinner to spin dry the residual liquid on the slide, use Cy3-labeled goat anti-mouse antibody (1:1000 dilution) as the secondary antibody, and incubate in a 37°C wet box for 45 minutes.

[0100] ④ Remove the slides and wash them three times with PBST, 5 min each time. Add DAPI (1:2000 dilution), incubate at room temperature for 15 min and wash as above.

[0101] ⑤ After drying, add anti-fluorescence quenching sealing agent in a dark place, cover with a coverslip, and store at 4℃ for later use.

[0102] ⑥Observe under fluorescence microscope.

[0103] Results: The flounder Muc5AC antibody specifically bound to the gill and skin mucin of the flounder, showing a positive fluorescence signal. The green fluorescence signal was observed to be scattered on the cell membrane surface, which was consistent with the fact that mucin was distributed in mucus cells and on the mucosal surface. Fig.10 ), while no fluorescence signal was observed in the negative control (NC).

[0104] Those skilled in the art will understand that within the protection scope of the present invention, modifications, additions and substitutions are possible for the above embodiments, and none of them exceeds the scope of protection requested by the present invention.

Claims

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

1.

2. A coupling protein, characterized in that The coupling protein is a molecule for enhancing immune effect coupled to the 5' end or 3' end of the antigen polypeptide according to claim 1; the molecule is a KLH carrier protein.

3. A specific antibody, characterized in that The specific antibody is prepared by immunizing Balb / c mice with the coupling protein described in claim 2 and collecting serum.

4. Use of the antibody according to claim 3 in preparing a reagent for identifying Muc5AC-positive mucous cells of Japanese flounder.

5. A kit for detecting or tracing Muc5AC-positive mucous cells of Japanese flounder, characterized in that: The kit comprises the antibody according to claim 3.

6. The kit according to claim 5, characterized in that The kit is a paraffin or frozen section detection kit.

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