An antigen epitope polypeptide and a paralichthys olivaceus bmp2 protein specific antibody prepared therefrom

Abstract

The application provides an antigen epitope polypeptide and a paralichthys dentatus BMP2 protein specific antibody prepared by the antigen epitope, designs an antigen epitope through bioinformatics analysis, adopts a segmented recombination expression strategy to obtain BMP2-N and BMP2-C recombination proteins in view of the low expression efficiency of full-length BMP2 protein in a prokaryotic system, and uses the recombination proteins as immunogens to prepare polyclonal antibodies. It is verified through immunoblotting and immunofluorescence that the prepared antibodies can specifically recognize the paralichthys dentatus BMP2 protein, have high affinity and good reaction activity, and effectively avoid the problems of false negative or low signal caused by insufficient cross reactivity of the antibodies. On this basis, a BMP2 immunological detection kit is constructed, precise positioning and expression analysis of the BMP2 protein in mucous membrane tissues such as skin, gill and intestinal tract of the paralichthys dentatus can be realized. The antibody and the kit provide a reliable tool for studying the role of BMP2 in fish mucosal immunity, and have important scientific research value and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biological products and fish immunology, specifically relating to an antigenic epitope polypeptide and its preparation in turbot ( Paralichthys olivaceus BMP2 protein-specific antibody. Background Technology

[0002] Fish live in aquatic environments rich in various microorganisms, and their bodies primarily rely on the mucosal immune system to defend against external pathogens. Mucosa-associated lymphoid tissue (MALT) is widely distributed in the gills, skin, and intestines—areas directly exposed to the external environment—and serves as a crucial barrier for sensing pathogenic stimuli and initiating an immune response. Turbot (Scourgette) Paralichthys olivaceus As an important marine aquaculture fish in my country, the fish is frequently infected by various pathogenic microorganisms during large-scale farming, leading to increased morbidity and significant economic losses. In recent years, research on fish mucosal immunity has gradually become a research hotspot in the field of aquatic disease control. In-depth analysis of its molecular regulatory mechanisms is of great significance for the development of new vaccines and immune enhancers.

[0003] Bone morphogenetic proteins (BMPs) are important members of the transforming growth factor-β (TGF-β) superfamily and were initially thought to primarily participate in developmental regulation. With further research, the functions of BMPs in the immune system have gradually gained attention. In innate immunity, BMP signaling participates in regulating the differentiation and functional state of myeloid cells such as macrophages, dendritic cells, and neutrophils; in adaptive immunity, they are closely related to the development and activation of T cells and B cells. As a key molecule in the BMP family, BMP2 plays an important role in the regulation of mucosal immunity in mammals. Existing studies have shown that BMP2 exhibits high expression levels in mucosal tissues such as the intestines and respiratory tract, participating in maintaining the structural and functional stability of the epithelial barrier; its expression level dynamically changes under inflammatory or infectious conditions and can influence the production of inflammatory factors by regulating signaling pathways such as NF-κB, exhibiting a bidirectional regulatory effect in different microenvironments. Furthermore, BMP2 is also considered an important molecular marker reflecting mucosal tissue remodeling under chronic inflammation. The above studies suggest that BMP2 plays an important role in maintaining mucosal homeostasis and regulating immune responses.

[0004] However, the immune regulatory mechanisms associated with BMP2 in lower vertebrates, especially bony fishes, lack systematic research. Existing studies largely focus on the transcriptional level, with insufficient understanding of its protein expression dynamics, cellular origin and distribution, and its relationship with mucus barrier function. Particularly, the specific regulatory role of BMP2 during pathogen infection remains unclear. One key factor contributing to this slow progress is the lack of specific detection tools for BMP2 in fish. While commercial antibodies against zebrafish BMP2 exist, their cross-reactivity in turbot has not been verified. Because zebrafish and turbot belong to different phylogenetic groups, their BMP2 protein sequences differ, and existing antibodies may not effectively recognize turbot BMP2, leading to unreliable results from immunoblotting, immunofluorescence, or immunohistochemistry.

[0005] More importantly, insufficient antibody specificity or poor affinity can easily lead to false negatives or significantly low signals during protein detection, thus masking the true expression level of the target protein. This severely affects the accuracy and reproducibility of experimental data and may even result in misjudgments of the biological function of BMP2. Furthermore, the low expression level and easy formation of inclusion bodies in full-length turbot BMP2 protein in prokaryotic expression systems further increase the technical difficulty of preparing high-quality specific antibodies.

[0006] Therefore, developing a highly sensitive antibody and its corresponding detection system capable of specifically recognizing the BMP2 protein in turbot is crucial for the accurate detection of BMP2 protein levels. This will not only help avoid false negatives or detection biases caused by antibody incompatibility, but also provide reliable technical support for a deeper understanding of the mechanism of action of BMP2 in fish mucosal immunity, and lay an important foundation for research related to aquatic disease control. Summary of the Invention

[0007] The purpose of this invention is to provide an antigenic epitope polypeptide and its preparation in turbot ( Paralichthys olivaceus The provided antibody is a BMP2 protein-specific antibody that can specifically bind to the BMP2 protein in turbot, enabling specific localization and tracing of the BMP2 protein in turbot mucosal tissue. This provides a key tool for in-depth research on the dynamic role of BMP2 in the immune response of turbot mucosa.

[0008] This invention first provides an antigen-peptide combination for preparing a specific antibody against turbot BMP2 protein, wherein the antigen-peptide combination comprises:

[0009] 1) BMP2-N polypeptide with the amino acid sequence SEQ ID NO:1:

[0010] VVPQYMVDLYRMHSANGDHSTKRPKSMGKHADRAASKANTIRSFHHEESMEALASLKGKTTQQFYFNLTSIPDEELITSAELRIYRDQVMGAATPNTSSRNSSTSDSGPTGGFHRINIYEIFRVPATNGREPLARLLDTRLVQDSLSRWESFDVSPAVSQWTSG (SEQ ID NO:1);

[0011] Its corresponding coding nucleotide sequence is as follows:

[0012] GTGGTGCCGCAGTACATGGTGGACCTTTACCGCATGCACTCAGCAAACGGAGACCACAGCACTAAACGGCCCAAGAGCATGGGGAAGCACGCAGATAGAGCCGCCAGCAAGGCCAACACGATTAGAAGCTTTCACCATGAAGAGTCTATGGAGGCCCTGGCCAGCCTGAAAGGCAAAACAACCCAGCAGTTCTACTTCAACCTCACTTCTATCCCTGATGAGGAGCTAATCACCTCTGCAGAGCTACGCATCTACAGGGATCAGGTTATGGGAGCAGCGACCCCGAACACCAGCTCCAGAAACAGCAGCACTAGCGATAGTGGTCCTACTGGTGGCTTCCATCGAATCAACATTTATGAGATATTCAGAGTTCCTGCCACTAATGGCAGGGAGCCTCTAGCACGTCTGCTGGACACTCGGCTAGTGCAGGACTCTTTAAGCCGCTGGGAGAGCTTTGATGTCAGCCCCGCTGTATCTCAGTGGACCTCCGGC(SEQ ID NO:2);

[0013] 2) BMP2-C polypeptide with an amino acid sequence of SEQ ID NO:3:

[0014] QWTSGKGHNHGFMVEVLHPDEGEMDGEHAKRRSKHVRVSRSLHQDQDSWPQARPLLVTYGHDGRGDSVLHTREKRQATLRKQRRKQQHKASCKRHALYVDFSDVGWNEWIVAPPGYHAFYCHGECPFPLADHLNSTNHAIVQTLVNSVNSNIPRACCVPTDLSPISLLYLDEYE;

[0015] The corresponding coding nucleotide sequence is as follows:

[0016] (SEQ ID NO:4);

[0017] This invention also provides an application of the aforementioned antigen-peptide combination in the preparation of polyclonal antibodies;

[0018] The present invention also provides a polyclonal antibody, which is prepared by purifying animal serum after immunizing animals with the above-mentioned antigen combination.

[0019] The immunized animal, as specifically described in the example, is a New Zealand white rabbit;

[0020] The present invention also provides the use of the polyclonal antibody in any of the following aspects:

[0021] 1) Application in the preparation of immunoassay reagents for detecting the expression level of BMP2 protein in turbot tissue samples;

[0022] 2) Application in the preparation of materials for studying the spatiotemporal expression and regulatory mechanism of BMP2 protein in turbot during bacterial infection;

[0023] 3) Application in the preparation of immunofluorescence detection reagents for tracing BMP2-positive cells in turbot mucosal tissue.

[0024] The present invention also provides an immunoassay kit for detecting or tracing turbot BMP2 protein in paraffin or frozen sections, the kit comprising the aforementioned polyclonal antibody.

[0025] The present invention has the following advantages:

[0026] 1) This invention uses bioinformatics methods to analyze the antigenic epitopes of turbot BMP2 protein. To address the technical problems of low expression efficiency and easy formation of inclusion bodies in the full-length protein in prokaryotic expression systems, a segmented recombination expression strategy is adopted to construct a truncated expression system. Combined with optimization of induction conditions and affinity chromatography purification, soluble recombinant antigen protein is successfully obtained, providing a reliable material basis for subsequent antibody preparation.

[0027] 2) The polyclonal antibody against turbot BMP2 prepared using the recombinant protein as an immunogen exhibits good specificity and high antigen-binding affinity. Immunoblotting and immunofluorescence experiments verified that this antibody can stably and specifically recognize turbot BMP2 protein, significantly reducing false negative results or low signal levels caused by insufficient antibody cross-reactivity or poor affinity, effectively improving the accuracy and repeatability of detection results.

[0028] 3) This invention constructs a turbot BMP2 immunoassay kit based on the aforementioned antibodies. This kit uses specific antibodies as its core component, combined with immunohistochemical detection methods, to achieve precise localization and expression analysis of BMP2 protein in the mucosal tissues of turbot, including skin, gills, and intestines. It is simple to operate, has high detection sensitivity, and is suitable for various experimental scenarios.

[0029] 4) Using the detection reagent system established in this invention, the dynamic expression of BMP2 in turbot during bacterial infection and immune response was systematically analyzed. The results showed that BMP2 exhibited specific regulatory changes in mucosal tissue, suggesting its involvement in the antibacterial immune process in fish. This invention provides a reliable technical means to elucidate the function of BMP2 in fish mucosal immunity.

[0030] 5) The specific antibody and matching kit prepared by this invention fill the gap in the detection tool for BMP2 protein level in turbot. It can not only be used for protein expression and localization analysis in basic research, but also has the potential for application in aquatic disease monitoring and immune regulation research. It has good scientific research value and industrialization prospects. Attached Figure Description

[0031] Figure 1 : A three-dimensional model of the BMP2 molecule in turbot; Figure 2 Phylogenetic tree diagram of the BMP2 molecule in turbot; Figure 3 Schematic diagram of the conserved domains and antigen fragment design of the turbot BMP2 molecule; Figure 4 SDS-PAGE analysis of recombinant proteins rBMP2-N and rBMP2-C, where M: protein molecular weight standard; lanes 1-3: rBMP2-N (1: whole bacterial protein before induction; 2: whole bacterial protein after IPTG induction; 3: purified protein); lanes 4-6: rBMP2-C (4: whole bacterial protein before induction; 5: whole bacterial protein after IPTG induction; 6: purified protein). Figure 5 : Immunoblotting assay to verify the specificity of the turbot BMP2 molecular antibody, where M: protein molecular weight standard; lanes 1-2: antibody recognition of purified recombinant protein (1: rBMP2-N (38.7 kDa); 2: rBMP2-C (40.3 kDa); lanes 3-4: antibody recognition of eukaryotic turbot BMP2 protein (3: negative control; 4: BMP2 (47.4 kDa)). Figure 6 Secondary mass spectrum of peptide 1 (DQVMGAATPNTSSR) as a result of liquid chromatography-tandem mass spectrometry verification of the specificity of the turbot BMP2 molecular antibody. Figure 7 : Secondary mass spectrum of peptide 2 (NSSTSDSGPTGGFHR) as a result of liquid chromatography-tandem mass spectrometry verification of the specificity of the turbot BMP2 molecular antibody; Figure 8 Secondary mass spectra of peptide 3 (IYRDQVMGAATPNTSSR) as a result of liquid chromatography-tandem mass spectrometry verification of the specificity of the turbot BMP2 molecular antibody. Figure 9 : Secondary mass spectrum of peptide 4 (SLHQDQDSWPQAR) as a result of liquid chromatography-tandem mass spectrometry verification of the specificity of the turbot BMP2 molecular antibody; in, Figures 6-9 The peaks marked in green correspond to the b-ion series, and the peaks marked in orange correspond to the y-ion series. Figure 10 Distribution of BMP2 molecules in the gills, skin, and intestines of turbot analyzed by indirect immunofluorescence (40 ×). The results show that BMP2-positive immunofluorescence signals (red) were detected in the gills, intestines, and skin. WGA (green) labeled mucin, and DAPI (blue) labeled cell nuclei. Control serum was used as a negative control instead of anti-BMP2 antibody on the far right. La, gill filaments; BC, gill cavity; Epi, epithelium; LP, lamina propria; Lu, intestinal lumen; IEC, intestinal epithelial cells. Scale bar: 50 μm. Detailed Implementation

[0032] The BMP2-N polypeptide used in this invention has an amino acid sequence corresponding to the peptide encoded by nucleotides 214 to 705 in the coding region of turbot BMP2 (Gene ID: 109632388), covering most of the TGF-β family propeptide domain. The BMP2-C polypeptide has an amino acid sequence corresponding to the peptide encoded by nucleotides 691 to 1212 in the coding region of turbot BMP2, covering the complete TGF-β superfamily mature peptide domain.

[0033] Polyclonal antibodies were prepared using BMP2-N and BMP2-C peptides as antigens. Indirect immunofluorescence assays confirmed that these antibodies specifically bound to the native BMP2 protein in the gills, skin, and intestinal mucosa of turbot. The results showed that specific positive signals were detected in the gills, skin, and intestines. This signal was clearly distributed in the epithelial layer of the mucosa and was visible on the outer surface of epithelial cells, appearing as punctate and filamentous patterns.

[0034] The present invention will now be described in detail with reference to the embodiments and accompanying drawings.

[0035] Example 1: Design of BMP2 antigen analysis for turbot

[0036] To design a highly specific antibody against turbot BMP2, a systematic bioinformatics analysis of its genes and proteins was first performed.

[0037] (1) Sequence acquisition and basic characteristic analysis

[0038] The BMP2 coding sequence of turbot (P. olivaceus) (Gene ID: 109632388, CDS: 1269 bp) was obtained from the NCBI database. SignalP-6.0 (https: / / services.healthtech.dtu.dk / services / SignalP-6.0 / ) predicted a 23-amino acid signal peptide at its N-terminus, exhibiting typical characteristics of a secreted protein. TMHMM (https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / ) analysis further confirmed the absence of a transmembrane domain. Domain analysis using the NCBI CDD and InterPro databases revealed two core domains: a TGF-β propeptide domain (amino acids 48-289) and a C-terminal TGF-β superfamily mature peptide domain (amino acids 322-422). Three-dimensional modeling of the mature peptide domain using AlphaFold2 revealed that the domain involves all seven conserved cysteine ​​residues and is surrounded by multiple β-sheets and α-helices. Figure 1 ).

[0039] (2) Evolutionary analysis and antigen fragment design

[0040] BMP2 protein sequences from different vertebrates were obtained from the NCBI and Ensembl databases, and a phylogenetic tree was constructed using MEGA software. The results showed that the BMP2 of turbot clustered with BMP2 of other bony fishes, with the BMP2 of turbot (P. olivaceus) and flounder (Hippoglossus hippoglussus) forming a separate terminal branch, confirming their close phylogenetic relationship and orthologous relationship. Figure 2 Based on the above comprehensive analysis, and considering the difficulty of expressing the full-length BMP2 protein in prokaryotic systems, this invention employs a segmented expression strategy for antigen design: after excluding the N-terminal signal peptide, two overlapping gene fragments are designed: the BMP2-N fragment (nucleotides 214-705 of the coding region), designed to cover the main part of the propeptide domain; and the BMP2-C fragment (nucleotides 691-1212 of the coding region), designed to completely cover the mature peptide domain containing all key functional residues. An overlapping sequence exists between the two fragments to ensure that potential conformational antigenic epitopes located at the fragment junction are not lost during immunization, thereby maximizing the diversity and integrity of antigenic epitopes. Figure 3 ).

[0041] Example 2: Cloning BMP2-N and BMP2-C peptide fragments

[0042] 1) Amplification of the gene core fragment

[0043] cDNA obtained by reverse transcription of total RNA from healthy turbot tissue was used as a template. Specific amplification primers were designed using Primer Premier 5.0 software. The primers were synthesized by Qingke Biotechnology Co., Ltd.

[0044] The primer sequences are as follows:

[0045] BMP2-NF: 5'-GTGGTGCCGCAGTACATGG-3'

[0046] BMP2-NR:5'-GCCGGAGGTCCACTGAGAT-3'

[0047] BMP2-CF: 5'-CAGTGGACCTCCGGCAAAG-3'

[0048] BMP2-CR: 5'-CTCATATTCATCCAGGTAGAGCAGG-3'

[0049] PCR reactions were performed using high-fidelity DNA polymerase.

[0050] Reaction system (25 μL): 2.5 μL 10× high-fidelity buffer, 2 μL dNTP Mixture, 1 μL each of forward and reverse primers (10 μM), 1 μL cDNA template, 0.5 μL high-fidelity DNA polymerase, and sterile ddH2O to 25 μL.

[0051] Reaction program: 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 60℃ annealing for 30 seconds, 72℃ extension for 60 seconds, for a total of 35 cycles; final extension at 72℃ for 5 minutes.

[0052] Take 5 μL of PCR product and perform 1% agarose gel electrophoresis at 180 V for 20 min. Cut out the target band of the correct size and purify and recover it using an agarose gel DNA recovery kit.

[0053] 2) Construction of recombinant cloning plasmids

[0054] The purified target fragment was ligated into the pClone007 Simple Vector.

[0055] Ligation system (10 μL): 1 μL carrier, 1 μL 10× Topo Mix, 1 μL target fragment, 7 μL DEPC water, react at 25 ℃ for 5 minutes.

[0056] The ligation product was transformed into DH5α competent cells, incubated on ice for 25 minutes, heat-shocked at 42 °C for 45 s, incubated on ice for 2 minutes, and then 600 μL of antibiotic-free LB liquid medium was added. The cells were then incubated at 37 °C for 60 minutes. 50 μL of the bacterial culture was spread onto LB agar plates containing 100 μg / mL ampicillin and incubated overnight at 37 °C.

[0057] Single clones were selected for colony PCR and identified using universal primers M13-F / R. The PCR reaction system and procedure were the same as before. After verification by agarose gel electrophoresis, positive clones were selected for sequencing. After confirming that the sequencing results matched the reference sequence, the positive bacterial culture was incubated in glycerol at -80℃ to obtain the cloning plasmids pClone007S T-BMP2-N and pClone007S T-BMP2-C for recombinant expression of BMP2-N and BMP2-C peptides.

[0058] Example 3: Recombinant expression preparation of BMP2-N and BMP2-C peptides

[0059] Using the sequencing-verified cloning plasmids pClone007S T-BMP2-N and pClone007S T-BMP2-C as templates, PCR amplification was performed using specific primers with homologous arms to obtain the target fragment that can be used for homologous recombination.

[0060] Primer design: A 15-20 bp homologous arm sequence (lowercase letter portion) homologous to the end of the linearized pET-32a(+) vector was added to the 5' end of the primers, using BamHI as the reference restriction site. The primers were synthesized by Qingke Biotechnology Co., Ltd.

[0061] The primer sequences are as follows:

[0062] BMP2-NF:

[0063] 5'-caaggccatggctgatatcggatccGTGGTGCCGCAGTACATGG-3'

[0064] BMP2-NR:

[0065] 5'-gcttgtcgacggagctcgaattGCCGGAGGTCCACTGAGAT-3'

[0066] BMP2-CF:

[0067] 5'-caaggccatggctgatatcggatccCAGTGGACCTCCGGCAAAG-3'

[0068] BMP2-CR:

[0069] 5'-gcttgtcgacggagctcgaattCTCATATTCATCCAGGTAGAGCAGG-3'

[0070] The PCR reaction system and procedure are the same as in Example 2. After purification and recovery of the PCR product, the target fragment with homologous arms is obtained.

[0071] ② The purified pET-32a(+) vector plasmid was digested with the restriction endonuclease BamHI at 37 ℃ for 1 h. The digestion system consisted of 1000 ng plasmid DNA, 0.5 μL BamHI, 5 μL Cutsmart, and sterile ddH2O to a final volume of 50 μL. After digestion, 1% agarose gel electrophoresis was performed for verification, and the linearized vector was recovered from the gel.

[0072] ③ Using homologous recombination cloning, purified BMP2-N and BMP2-C PCR fragments were mixed with linearized pET-32a(+) vector, respectively. A seamless cloning enzyme was used, with a fragment-to-vector molar ratio of 3:1, and the mixture was reacted at 37 ℃ for 60 min. 5 μL of the recombinant product was transformed into 100 μL of chemically competent *E. coli* DH5α cells, incubated on ice for 30 min, heat-shocked at 42 ℃ for 45 s, immediately incubated on ice for 2 min, and then 600 μL of antibiotic-free LB liquid medium was added. The cells were then incubated at 37 ℃ with shaking at 200 rpm for 60 min to recover. 50 μL of the bacterial culture was spread onto LB agar plates containing 100 μg / mL ampicillin and incubated upside down at 37 ℃ for 12–16 h.

[0073] ④ The following day, single colonies were picked and dissolved in 20 μL of sterile water as templates. Preliminary screening was performed using universal vector primers (T7 promoter / terminator primers). Positive clones were sent to a sequencing company for sequencing. The returned sequencing results were compared with the original reference sequence using the BLASTN tool on the NCBI website, confirming 100% nucleotide sequence identity and correct reading frames with no frameshift mutations. Thus, recombinant plasmids pET-32a-BMP2-N and pET-32a-BMP2-C were successfully constructed.

[0074] (2) Protein-induced expression

[0075] Prepare the buffer solution required for protein purification:

[0076] Equilibration buffer: Na₂HPO₄ 14.5 g; NaH₂PO₄ 1.48 g; NaCl 29.3 g; urea 480 g; imidazole 40 mM (2.72 g); adjust pH to 7.4, and bring ultrapure water to a final volume of 1 L. Filter through a 0.45 μm membrane.

[0077] Elution buffer: Na₂HPO₄ 14.5 g; NaH₂PO₄ 1.48 g; NaCl 29.3 g; urea 480 g; imidazole 500 mM (34 g); pH adjusted to 7.4, ultrapure water brought to 1 L. Filtered through a 0.45 μm membrane.

[0078] Protein dialysis buffer: Na₂HPO₄ 17.91 g; NaH₂PO₄ 1.31 g; NaCl 2.93 g; EDTA 0.37 g; glycerol 50 mL; glycine 10 g; urea (0 M, 2 M, 4 M, 6 M, 8 M). Adjust pH to 7.4, and bring volume to 1 L with ultrapure water. Filter through a 0.45 μm membrane.

[0079] ① The recombinant plasmids pET-32a-BMP2-N and pET-32a-BMP2-C, verified by sequencing, were heat-shocked and transformed into chemocompetent cells of *Escherichia coli* BL21(DE3). After plating and culturing, single positive colonies were picked and inoculated into 5 mL of LB broth containing 100 μg / mL ampicillin, and cultured overnight at 37 ℃ with shaking at 220 rpm. The bacterial culture was then transferred to 50 mL of fresh LB resistant medium at a 1:100 inoculation ratio and cultured until the OD600 was approximately 0.6-0.8. At this point, isopropyl-β-D-thiogalactoside was added to the culture to a final concentration of 0.5 mM. The culture flasks were then transferred to a shaker at 37 ℃ and expression was induced for another 12 h with shaking at 220 rpm.

[0080] ② After induction, take 1 mL of bacterial culture before and after induction, centrifuge at 8000 g for 2 minutes to collect the bacterial cells. Wash three times with sterile PBS, resuspend in 100 μL of loading buffer, and denature in a metal bath at 100 ℃ for 10 min. Take 10-20 μL of sample for 12% SDS-PAGE gel electrophoresis (20 mA stacking gel, 30 mA separating gel). After Coomassie Brilliant Blue R-250 staining, compared with the sample before induction, significantly thickened and enhanced protein bands were observed at approximately 38.7 kDa (BMP2-N) and 40.3 kDa (BMP2-C), indicating successful expression of the recombinant protein. Figure 4 500 mL of the induced bacterial culture was aliquoted into 50 mL centrifuge tubes and centrifuged at 8000 g for 5 minutes at 4 °C. The culture was concentrated to the bottom of the 50 mL tube, resuspended in equilibration buffer, and then sonicated at 300 W with 50% amplitude, sonicating for 3 seconds and pausing for 3 seconds until the bacterial culture was clear and the graduations on the beaker wall were visible. The culture was then centrifuged at 12000 g for 10 minutes, and the supernatant was collected and filtered through a 0.45 μm pore size syringe filter.

[0081] ③ Perform nickel affinity chromatography using a protein purification system. Equilibrate the HisTrap HP nickel column with denaturing binding buffer, and then load the filtrate. After loading, wash with 15 column volumes of denaturing wash buffer until the UV absorption baseline stabilizes. Finally, perform staged elution with denaturing elution buffer and collect the solution corresponding to the elution peak.

[0082] (3) Dialysis refolding and preparation of end products

[0083] The eluent containing the target protein was placed into a dialysis bag with a molecular weight cutoff of 10 kDa. Dialysis was performed sequentially in dialysis buffers (based on PBS, pH 7.4) with varying urea concentrations at 4 °C under magnetic stirring: 8 M, 6 M, 4 M, 2 M, and 0 M urea overnight. Finally, dialysis was performed overnight in PBS and pure water to completely remove urea. A small amount of the renatured protein solution was taken and the protein concentration was determined using the BCA method. The remaining solution was aliquoted into cryovials and pre-frozen at -80 °C for 12 h. Then, it was transferred to a vacuum freeze dryer and freeze-dried for 36–48 h at a temperature below -50 °C and a vacuum level below 10 Pa until a white, fluffy, porous solid was obtained, thus yielding the recombinant protein lyophilized powder.

[0084] Example 4: Preparation of polyclonal antibody against turbot BMP2

[0085] rBMP2-N and rBMP2-C protein powders were dissolved and quantified separately in sterile PBS, and then mixed in equimolar proportions to serve as immunogens. Before the first immunization, approximately 2 mL of whole blood was collected from the marginal ear vein of experimental New Zealand white rabbits. After standing at room temperature for 2 h, the blood was incubated overnight at 4 ℃, and the serum was separated by centrifugation at 3000 g for 10 minutes. This serum was aliquoted as a negative control and stored at -80 ℃.

[0086] Take 400 μg of the mixed immunogen (dissolved in 200 μL PBS) and draw it into two separate syringes connected to a communicating vessel, along with an equal volume (200 μL) of Freund's complete adjuvant. Slowly and repeatedly push the plunger to mix the two liquids until a stable water-in-oil emulsion is formed, in which droplets do not spread on the water surface for 30 seconds. For booster immunization, use an equal volume of Freund's incomplete adjuvant to emulsify the immunogen.

[0087] Multiple subcutaneous injections were administered on the back. The fur on the rabbit's back was shaved and disinfected. Using a 1 mL syringe, the emulsified antigen was drawn up and injected subcutaneously at at least four points on the back, with approximately 100 μL injected at each point (total 400 μL / rabbit). During injection, the needle was inserted obliquely into the subcutaneous tissue, held for a moment after injection, and then slowly rotated out to prevent leakage.

[0088] The immunization schedule is as follows:

[0089] (1) Day 0 (initial immunization): CFA emulsion antigen.

[0090] (2) Days 14, 28, 42, and 56 (first to fourth booster immunizations): IFA emulsified antigen.

[0091] (3) Day 70 (fifth booster immunization, final): IFA emulsified antigen.

[0092] Before each immunization, observe the rabbits' mental state, appetite, and injection site reaction. On day 80 (10 days after the last immunization), euthanize the rabbits by exsanguination through the carotid artery, collecting whole blood into 50 mL sterile centrifuge tubes. Incubate the blood at an angle in a 37°C oven for 1 hour, then transfer it to a 4°C refrigerator and let it stand overnight to allow the blood clots to fully shrink. The next day, gently separate the blood clots from the tube wall with a sterile bamboo skewer and centrifuge at 3000 g for 20 minutes at 4°C. Carefully aspirate the clear, pale yellow serum from the supernatant using a sterile pipette, taking care to avoid aspirating red blood cells. Aliquot the serum into sterile centrifuge tubes and store at -80°C.

[0093] Example 5: Immunoblot identification of antibody specificity and reactivity

[0094] To assess antibody specificity, its reactivity against recombinant and ectopic expressed proteins was validated using Western blotting.

[0095] (1) Validation of recombinant protein

[0096] ① Mix 1 μg of purified recombinant protein with loading buffer and denature in a metal bath at 100 °C for 10 minutes. Separate the protein by 12% SDS-PAGE electrophoresis, using a stacking gel at a constant current of 20 mA and a separating gel at a constant current of 30 mA.

[0097] ② After electrophoresis, the gel, along with a PVDF membrane (0.45 μm pore size) pre-activated with methanol for 1 minute, filter paper, and a sponge pad, were assembled into a "sandwich" structure in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol). The membrane was then transferred to the PVDF membrane at a constant current of 400 mA for 10 minutes at room temperature using a rapid transfer buffer.

[0098] ③ After the transfer is complete, remove the PVDF membrane and immerse it in the rapid blocking buffer for 1 hour at room temperature.

[0099] ④ Discard the blocking solution, add 1:2000 diluted polyclonal antibody for preparing turbot BMP2, ensuring complete coverage. Incubate at room temperature for 1.5 h, then wash three times with PBST for 5 min each time.

[0100] ⑤ Discard excess PBST, add HRP-labeled goat anti-rabbit IgG secondary antibody diluted 1:20000, and incubate at room temperature for 45 minutes. Wash three times with PBST, 5 minutes each time.

[0101] ⑥ Add an enhanced chemiluminescent substrate and image protein bands using a chemical imaging system.

[0102] (2) Verification of eukaryotic proteins

[0103] ① HEK293T cells were transfected with pTag-HA-BMP2 plasmid and empty pTag-HA vector (negative control) using Lipo8000 transfection reagent. 48 h after transfection, cells were lysed with RIPA lysis buffer containing PMSF.

[0104] ②The lysates from the BMP2 transfection group and the empty vector control group were then incubated independently with anti-HA magnetic beads under the same conditions to enrich HA-tagged proteins.

[0105] ③ The enriched samples were separated by SDS-PAGE, and the subsequent immunoblotting and detection procedures were exactly the same as those for the recombinant protein verification described above.

[0106] The results showed specific bands at the corresponding positions of recombinant rBMP2-N (38.7 kDa) and rBMP2-C (40.3 kDa) proteins, as well as eukaryotic protein from cell lysates (47.4 kDa), while no color signal was observed in the negative control. Figure 5 This indicates that the prepared antibody has high specificity for turbot BMP2.

[0107] Example 6: Mass spectrometry validation of antibody target specificity

[0108] To confirm the target protein recognized by the antibody at the sequence level, the present invention performs mass spectrometry analysis on the affinity-purified target protein complex.

[0109] The sample preparation procedure was consistent with the eukaryotic expression validation experiment. The loaded sample and negative control sample were separated by SDS-PAGE in parallel lanes of the same gel. The gel was stained with Coomassie Brilliant Blue and then destained using a destaining solution (10% acetic acid, 40% methanol). Coomassie Brilliant Blue bands corresponding to the positive signal positions in the Western blotting (approximately 47.4 kDa) were precisely excised from the gel for mass spectrometry identification and sent to Shanghai Sangon Biotech Co., Ltd. for liquid chromatography-tandem mass spectrometry (LC-MS / MS) identification. Raw mass spectrometry data were processed using MaxQuant software (v2.2.0.0) and its built-in Andromeda search engine. The search was performed against a turbot (P. olivaceus) protein database. Mass spectrometry analysis revealed four BMP2-specific peptides with a confidence score of 98.58. Furthermore, absolute quantification based on iBAQ values ​​showed that BMP2 accounted for approximately 66% of the total protein content, indicating that the prepared antibody has high specificity for turbot BMP2.

[0110] Example 7: Immunofluorescence identification of BMP2 tissue localization

[0111] To verify the binding ability of the antibody to BMP2 in natural tissues, immunofluorescence staining was performed on frozen sections of gills, skin, and intestinal mucosa from healthy turbot.

[0112] ① Frozen sections of mucosal tissue, gills, intestines, and skin from healthy turbot were placed at room temperature for 30 minutes to thaw. They were then rinsed three times with PBS for 5 minutes each time.

[0113] ② Permeate with PBS containing 0.1% Triton X-100 at room temperature for 15 min, then add 5% BSA (dissolved in PBS) and block at 37°C in the dark for 45 min.

[0114] ③ Discard the blocking solution, and add a 1:500 diluted turbot BMP2 polyclonal antibody to the tissue, ensuring the liquid completely covers the tissue. Incubate at 37 ℃ in the dark for 1.5 h, and wash three times with PBS for 5 minutes each time.

[0115] ④ Discard excess PBS, add Alexa Fluor 649-labeled goat anti-rabbit IgG secondary antibody diluted 1:2000, and simultaneously add FITC-labeled WGA (1:500) to label the mucus. Incubate at 37°C in the dark for 45 minutes. Wash three times with PBS, 5 minutes each time.

[0116] ⑤ Discard excess PBS, add 1:1000 diluted DAPI to stain cell nuclei for 10 minutes, wash 3 times with PBS, and then add anti-fluorescence quenching mounting solution to mount the slide.

[0117] ⑥ Observe and acquire images under a fluorescence microscope.

[0118] The results showed that BMP2-specific red fluorescence signals were detected in the gills, hindgut, and skin. Histological localization results showed that the signal was clearly distributed in the cytoplasm of mucosal epithelial cells and was visible on the outer surface of epithelial cells, appearing as punctate and filamentous distributions. In gill tissue, specific fluorescence was visible at the edge of the gill lamellae (La); in skin tissue, the fluorescence signal was localized in the epidermis (Epi); in intestinal tissue, the positive signal mainly appeared in the epithelial layer (IEC) on the luminal side of the intestine. Compared with the negative control (using non-immune serum instead of the primary antibody), the signal was specific and the background was clean. These results directly confirm that the antibody of this invention can be used in various mucosal tissues and can accurately trace the in situ distribution of BMP2 protein. Figure 10 ).

[0119] Example 8: Composition and Use of the Detection Kit

[0120] The standardized test kit of the present invention comprises the following components: antigen retrieval solution, blocking solution, anti-turbot BMP2 polyclonal antibody, Alexa Fluor 649 labeled goat anti-rabbit IgG antibody, DAPI nuclear staining solution, PBS buffer (10×) and anti-fluorescence quenching mounting medium, and is accompanied by detailed instructions.

[0121] Typical procedures include: antigen retrieval from tissue sections, BSA blocking, sequential incubation with primary and secondary antibodies, DAPI counterstaining, mounting, and observation under a fluorescence microscope. This kit standardizes the BMP2 detection process, facilitating widespread application and making it particularly suitable for immunological research scenarios.

[0122] In summary, this invention successfully prepared a specific polyclonal antibody against turbot bone morphogenetic protein 2 (BMP2). This antibody can serve as a molecular probe for recognizing BMP2 protein, enabling protein-level detection through immunoblotting, immunofluorescence, and other methods. It allows for the specific localization and tracing of BMP2 protein in turbot mucosal tissues (especially gills, skin, and intestines), providing a crucial tool for in-depth research into the dynamic role of BMP2 in the turbot mucosal immune response.

[0123] The development of this antibody fills the technological gap in the detection tool for BMP2 protein in turbot, and can provide important support for revealing the evolutionary conservation and functional mechanism of BMP2 in the mucosal immunity of bony fish. It also provides an innovative solution for the disease prevention and control needs of aquaculture based on the assessment of mucosal immune status, and lays the tool foundation for the green and healthy development of aquaculture.

[0124] Those skilled in the art should understand that any equivalent substitutions or modifications made to the specific parameters, steps, or reagents in the above embodiments without departing from the concept of the present invention fall within the protection scope defined by the claims of the present invention.

Claims

1. An antigenic epitope polypeptide, characterized in that, The antigenic epitope polypeptide comprises a polypeptide with the amino acid sequence SEQ ID NO:1 and a polypeptide with the amino acid sequence SEQ ID NO:

3.

2. The antigenic epitope polypeptide according to claim 1, characterized in that, The polypeptide with the amino acid sequence of SEQ ID NO:1 encodes a nucleotide sequence of SEQ ID NO:

2.

3. The antigenic epitope polypeptide according to claim 1, characterized in that, The polypeptide with the amino acid sequence SEQ ID NO:3 encodes a nucleotide sequence SEQ ID NO:

4.

4. The use of the antigenic epitope polypeptide according to claim 1 in the preparation of polyclonal antibodies.

5. A polyclonal antibody, wherein the polyclonal antibody is prepared by purifying animal serum after immunizing an animal with the antigenic epitope polypeptide of claim 1.

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

7. Use of the polyclonal antibody of claim 5 in the preparation of articles for detecting or tracing BMP2 protein.

8. An article for detecting or tracing BMP2 protein, characterized in that, The product contains the polyclonal antibody as described in claim 5.

9. The article of claim 8, characterized in that, The product is an immunoassay kit for detecting or tracing turbot BMP2 protein in paraffin or frozen sections.

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