Antibacterial peptide troleap2-21 and its use in the preparation of products against bacteria and to maintain the integrity of the fish tissues

Abstract

The present application relates to antibacterial peptide TroLEAP2-21 and its application in the preparation of antibacterial and maintaining fish tissue integrity products, belong to the technical field of biological polypeptide, the amino acid sequence of the antibacterial peptide is shown as SEQ ID NO.1.The present application also provides the application of the antibacterial peptide TroLEAP2-21 of the Trachurus japonicus in the preparation of antibacterial products, the bacteria are Lactococcus garviae and Staphylococcus epidermidis.The antibacterial peptide TroLEAP2-21 can change the permeability of bacterial cell membrane, induce the depolarization of bacterial cell membrane, degrade bacterial genomic DNA, and cause the morphological change of bacteria, thereby playing an antibacterial role.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide technology, and specifically discloses an antimicrobial peptide TroLEAP2-21 and its application in the preparation of antimicrobial products and products that maintain the integrity of fish tissues. Background Technology

[0002] Antimicrobial peptides, serving as the first line of defense in the innate immune system, are a class of small molecule polypeptides widely found in all organisms, including fish. Research on fish antimicrobial peptides, a crucial direction in addressing the global antibiotic resistance crisis, has evolved from early species resource screening to a stage that emphasizes both mechanism analysis and application development. Fish, due to their continuous exposure to rich microbial communities in their aquatic environment, have evolved a unique innate immune system, in which antimicrobial peptides, as key effector molecules, exhibit remarkable diversity. Currently, several antimicrobial peptide families, including hepcidins, defensins, cathelicidins, and histone-derived peptides, have been identified in economically important fish species such as Salmoniformes, Perciformes, and Tetraodonta. These molecules not only possess characteristic structures such as α-helices and β-sheets but also exert broad-spectrum antimicrobial activity through multiple mechanisms, including membrane disruption, intracellular targeting (interfering with nucleic acid / enzyme function), and immune regulation (chemotaxis of immune cells, regulation of inflammatory factors). In recent years, with the development of transcriptomics and proteomics technologies, researchers have been continuously discovering novel antimicrobial peptides from pathogen-contact tissues such as fish mucus, skin, gills, and intestines through a strategy combining bioinformatics mining and functional verification. The expression of these antimicrobial peptides is strongly induced by pathogen-associated molecular patterns (PAMPs), such as LPS and peptidoglycan, and activates the NF-κB signaling pathway through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), thereby initiating the transcriptional expression of antimicrobial peptide genes. This rapid response mechanism is a key immune strategy for fish to cope with sudden infections and also provides a theoretical basis for the development of novel immune enhancers. Summary of the Invention

[0003] The technical problem to be solved by this invention is to provide the application of TroLEAP2-21, an antimicrobial peptide from oval pomfret, in the preparation of antimicrobial products. The antimicrobial peptide exhibits antimicrobial activity against Gram-positive bacteria (Lactococcus garvieae and Staphylococcus epidermidis) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi).

[0004] The technical solution of the present invention is as follows:

[0005] The antimicrobial peptide TroLEAP2-21 from the oval pomfret has the following amino acid sequence: KRFFAQRKTAVALCIVLLMLA (SEQ ID NO.1).

[0006] The application of TroLEAP2-21, an antimicrobial peptide from oval pomfret, in the preparation of antimicrobial products, wherein the bacteria are Lactococcus gasseri, Staphylococcus epidermidis, Vibrio alginolyticus, and / or Vibrio harveyi.

[0007] Furthermore, the antimicrobial peptide TroLEAP2-21 can alter the permeability of bacterial cell membranes, induce bacterial cell membrane depolarization, degrade bacterial genomic DNA, and cause morphological changes in bacteria.

[0008] Application of TroLEAP2-21, an antimicrobial peptide from oval pomfret, in the preparation of drugs for maintaining the integrity of fish tissue structure, wherein the application is to use the antimicrobial peptide TroLEAP2-21 to repair damage to oval pomfret tissue.

[0009] The beneficial effects of this invention compared to the prior art: In the prior art (Lei Y, Qiu R, Shen Y, Zhou Y, Cao Z, Sun Y. Molecular characterization and antibacterial immunity functional analysis of liver-expressed antimic robial peptide 2 (LEAP-2) gene in golden pompano (Trachinotus ovatus). Fish Shellfish Immunol. 2020 Nov;106: 833-843. doi: 10.1016 / j.fsi.2020.09.002. Epub 2020 Sep 3. PMID: 32891790. and CN110551732A;2019.12.10 The antibacterial effects of LEAP2 protein against Edwardsiella tarda, Streptococcus agalactiae, Gram-positive bacteria (Streptococcus agalactiae, Staphylococcus aureus, Bacillus), Gram-negative bacteria (Vibrio alginolyticus, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio anguillarum, Brilliantobacterium melanogaster, Escherichia coli), and fungi (yeast) are described in CN110551732A. Lei Y et al. disclosed that the LEAP-2 gene exerts its antibacterial effect by overexpressing and affecting the expression of related immune regulatory genes. CN110551732A discloses that a 106-amino acid peptide has antibacterial activity against Brilliantobacterium melanogaster, Vibrio anguillarum, Streptococcus agalactiae, and Bacillus.

[0010] The present invention relates to a novel antimicrobial peptide, TroLEAP2-21, identified from the oval pomfret (Trachinotus ovatus). This peptide consists of only 21 amino acids, and the sequence of the peptide is not in the middle of the 106 amino acids disclosed in CN110551732A. Therefore, the polypeptide of the present invention was not disclosed in CN110551732A.

[0011] The 21 amino acids of the polypeptide described in this invention form a distinct amphiphilic α-helix structure, with positively charged hydrophilic residues (such as K and R) concentrated on one side, and a hydrophobic surface formed by nonpolar hydrophobic residues (such as A, V, L, I, and F) on the other side. This structure is key to its interaction with bacterial membranes and the generation of antibacterial activity. This invention systematically evaluated its antibacterial activity against Gram-positive bacteria (Lactococcus garvieae and Staphylococcus epidermidis) and Gram-negative bacteria (Vibrio alginolyticus and Vibrio harveyi). Antibacterial efficacy experiments showed that TroLEAP2-21 exhibited significant inhibitory effects against all four pathogens, with a half-maximal inhibitory concentration (MIC50) of 60 μM. Transmission electron microscopy revealed significant morphological and structural damage in bacterial cells treated with TroLEAP2-21. Propidium iodide (PI) staining further confirmed that this peptide significantly increases bacterial cell membrane permeability. Membrane potential assays showed that TroLEAP2-21 induced significant cell membrane depolarization in four bacterial species. Furthermore, except for Vibrio alginolyticus, the peptide exhibited genomic DNA binding and degradation activity against the other three bacteria. In vivo experiments confirmed that TroLEAP2-21 significantly improved the survival rate of infected pomfret and reduced histopathological damage, demonstrating optimal therapeutic efficacy. Attached Figure Description

[0012] Figure 1 shows the high-performance liquid chromatography (HPLC) and mass spectrum (MS) of the chemically synthesized TroLEAP2-21; A is the chromatogram, and B is the mass spectrum.

[0013] Figure 2 shows the three-dimensional structure prediction of TroLEAP2-21;

[0014] Figure 3 The agglutination effect of TroLEAP2-21 peptide on four bacteria (Staphylococcus epidermidis, Lactococcus garvieae, Vibrio harveyi, and Vibrio alginolyticus) under different treatment conditions is shown; A, B, and C represent the effects of TroLEAP2-21, TroLEAP2-21+Ca, etc., respectively. 2+ and TroLEAP2-21+Ca 2+ +EDTA treatment L.garvieae The diagram shows the changes in bacteria; D, E, and F represent the results of using TroLEAP2-21 and TroLEAP2-21+Ca, respectively. 2+ and TroLEAP2-21+Ca 2++EDTA processing .epidermidis Changes in bacteria; G, H, and I represent the effects of TroLEAP2-21 and TroLEAP2-21+Ca, respectively. 2+ and TroLEAP2-21+Ca 2+ +EDTA treatment V. alginolyticus Changes in bacteria; J, K, and L represent the effects of TroLEAP2-21 and TroLEAP2-21+Ca, respectively. 2+ and TroLEAP2-21+Ca 2+ +EDTA treatment V.harveyi Changes in bacteria;

[0015] Figure 4 Evaluation of the antibacterial effect of TroLEAP2-21 peptide against pathogens, where A represents the antibacterial effect of different concentrations of TroLEAP2-21 against pathogens. L. garvieae The antibacterial effect diagram shows that B represents the effects of different concentrations of TroLEAP2-21 on the antibacterial activity of the drug. S. epidermidis The antibacterial effect diagram shows that C represents different concentrations of TroLEAP2-21 against [the bacteria / the virus]. V. alginolyticus The antibacterial effect diagram shows that D represents the effects of different concentrations of TroLEAP2-21 on the antibacterial activity of the drug. V.harveyi The antibacterial effect diagram;

[0016] Figure 5 The effect of TroLEAP2-21 on bacterial cell membrane permeability is shown in the image on the left. The top horizontal line represents the control group and different concentrations of the four bacteria in the row. The images of the four bacteria on the left are the experimental results of the bacteria at the corresponding TroLEAP2-21 concentration in the top horizontal line.

[0017] Figure 6 TroLEAP2-21's depolarizing effect on the cell membrane of tested bacteria, where A is... L. garvieae B is S. epidermidis C is V. alginolyticus D is V.harveyi ;

[0018] Figure 7 The effect of TroLEAP2-21 on bacterial genomic DNA, where A represents... L. garvieae B is S. epidermidis C is V. alginolyticus D is V.harveyi ;

[0019] Figure 8To illustrate the effect of TroLEAP2-21 on bacterial cell morphology, the left vertical axis shows the treatment groups with and without TroLEAP2-21, and the top horizontal axis shows different bacteria. Each figure represents the experimental results for the corresponding treatment on the left and the bacteria above it.

[0020] Figure 9 The images show pathological analysis of gill, liver, and intestinal tissues of fish infected with Lactococcus gasseri and Vibrio harveyi after treatment with phosphate-buffered saline (PBS) or TroLEAP2-21 peptide. A is an experimental image of Lactococcus gasseri infection, and B is an experimental image of Vibrio harveyi infection. Each image represents the experimental results of the treatment on the left for the corresponding tissue above the image.

[0021] Figure 10 for L. garvieae and V. harveyi The effect of TroLEAP2-21 treatment on individual survival rate after infection is shown in the figure, where A represents... L. garvieae, B is V. harveyi ;

[0022] Figure 11 Detection of hemolytic reaction of cells by TroLEAP2-21 oval pomfret. Detailed Implementation

[0023] The technical solution of the present invention will be further studied through the following embodiments, but the scope of protection of the present invention is not limited in any way by the embodiments.

[0024] Example 1: Preparation process of antimicrobial peptide TroLEAP2-21

[0025] 1.1 Core Region Screening and Design: Based on the amino acid sequence characteristics, secondary structure, peptide length and physicochemical properties of the full-length protein, the potential antibacterial core region sequence of TroLEAP2-21 (KRFFAQRKTAVALCIVLLMLA, SEQ ID NO.1) was screened.

[0026] 1.2 Chemical Synthesis: The selected peptide sequence was chemically synthesized by Sangon Biotech Co., Ltd. Solid-phase peptide synthesis (SPPS) was employed, with amidation modification at the carboxyl terminus (C-terminus). This modification helps improve the stability and bioactivity of the peptide. The crude product obtained was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to achieve a purity of over 95%. The purified peptide was identified using mass spectrometry (LCMS-2010A, Shimadzu Corporation, Japan). Figure 1As shown, chromatogram A shows main peaks at 6.340 min and 6.933 min, indicating high peptide purity. Mass spectrum B confirms the molecular weight of the TroLEAP2-21 peptide, with the main peak located at m / z 598.85 ([M+4H]). 4+ ), 798.65 ([M+3H]) 3+ ) and 1196.80 ([M+2H] 2+ These charge states correspond to a calculated molecular weight of approximately 2395 Da. This indicates that the expected target peptide was obtained with high purity. The molecular weight was confirmed to be consistent with the theoretical value, thus verifying the peptide's identity and the accuracy of its synthesis.

[0027] 1.3 Structural characteristics of TroLEAP2-21: Sequence: KRFFAQRKTAVALCIVLLMLA (SEQ ID NO. 1); Length: 21 amino acid residues; Net charge: +4 (strongly positively charged at physiological pH, facilitating binding to negatively charged bacterial membranes), contributed by 2 lysines (Lys, K) and 2 arginines (Arg, R); Hydrophobicity: 69% (highly hydrophobic, facilitating interaction with the bacterial membrane phospholipid bilayer); Hydrophobic moment: 0.155 (an indicator of amphiphilicity); Special residues: Contains 1 proline (Pro, P) and no cysteine ​​(Cys, C).

[0028] Generate and visualize using Swiss-Model and Pymol software, such as Figure 2 As shown, the model further illustrates the possible α-helical structure that TroLEAP2-21 can form in three-dimensional space. The spatial distribution of peptides forming α-helices is illustrated. As shown, TroLEAP2-21 can form a distinct amphiphilic α-helical structure, with one side concentrated with positively charged hydrophilic residues (such as K and R), while the other side consists of a hydrophobic surface formed by nonpolar hydrophobic residues (such as A, V, L, I, and F). This structure is key to its interaction with bacterial membranes and the generation of antibacterial activity.

[0029] Example 2:

[0030] 1. Bacterial Aggregation Assay: The bacterial aggregation assay was performed using a modified technique based on the research of Duan et al. (Z. Duan, F. Zhang, X. Wang, H. Li, D. Zhou, Q. Chen, Z. Tao, Z. Chen, G. Yu, H. Yu, C-typelectin 12B / 4E of black rockfish (Sebastes schlegelii) macrophages as pattern recognition receptors in the antibacterial mechanism of exploration, Fish & Shellfish Immunology 150 (2024) 109636.). The bacterial suspension was washed twice with PBS buffer, then resuspended and diluted in PBS to a final concentration of 1 × 10⁻⁶. 8 CFU / mL. Take 100 μL of each bacterial suspension and mix it with an equal volume of TroLEAP2-21 peptide alone, peptide plus Ca. 2+ And peptides with the addition of Ca 2+ Mix with EDTA (final peptide concentration 15 μM). Incubate the mixture at 24°C under static conditions for 30 minutes. After incubation, observe the sample under a microscope to assess whether any form of bacterial aggregation is observed.

[0031] Observations show that in Ca 2+ In its presence, TroLEAP2-21 can significantly agglutinate Gram-positive bacteria ( L. garvieae , S. epidermidis ) and Gram-negative bacteria ( V. alginolyticus , V. harveyi And through EDTA chelation of Ca 2+ Subsequently, the bacterial agglutination activity of the peptide was completely lost. Figure 3 ).

[0032] 2. Antibacterial activity of TroLEAP2-21: To investigate whether TroLEAP2-21 can inhibit bacterial growth, an inhibition zone experiment was conducted (Y. Chen, L. Yao, Y. Wang, X. Ji, Z. Gao, S. Zhang, G. Ji, Identification of ribosomal

[0033] Protein L30 as an uncharacterized antimicrobial protein, Developmental Comparative Immunology 120 (2021) 104067., with slight modifications. Bacterial cells were washed three times with PBS buffer and then normalized to a final concentration of 1 × 10⁻⁶. 5 CFU / mL. Bacterial cultures were exposed to four different concentrations of TroLEAP2-21 peptide (0 μM, 15 μM, 30 μM, and 60 μM) and incubated at ambient temperature for 1 hour. Then, 20 μL of each bacterial solution was spread onto solid culture plates and incubated overnight at 37°C. The bacterial growth inhibition rate was calculated using the following formula: [(Control group colony count - Experimental group colony count) / Control group colony count] × 100.

[0034] Antibacterial efficacy test results showed that TroLEAP2-21 was effective against Gram-positive bacteria ( L. garvieae , S. epidermidis ) and Gram-negative bacteria ( V. alginolyticus , V. harveyi All four bacteria exhibited antibacterial activity. The results showed that TroLEAP2-21 demonstrated significant antibacterial activity against all four bacteria, with a half-maximal inhibitory concentration (MIC) of [value missing]. 50 The value is 60 μM ( Figure 4 ).

[0035] 3. TroLEAP2-21 alters bacterial cell membrane permeability: Flow cytometry was used to investigate the effect of TroLEAP2-21 on membrane permeability, following a previous study by Ali et al. (W. Ali, Y. Chen, Z. Wang, M. Sun, Y. Song, X. Guo, X. Wang, Y. He, J. Qi, Evaluating the Antimicrobial Efficacy of TroLEAP2 like-27 peptide in golden pompano (Trachinotus ovatus) against Bacterial Pathogens, Fish & Shellfish Immunology 162 (2025) 110310.). In short, bacterial cultures were diluted with phosphate-buffered saline (PBS) to a cell density of 1 × 10⁻⁶ cells / mL. 7CFU / mL. Subsequently, 300 μL of bacterial suspension was mixed with 300 μL of TroLEAP2-21 to achieve the final concentrations (0, 3.75 μM, 7.5 μM, 15 μM). After adding the peptide, the samples were incubated at room temperature for one hour. 200 μL of PI dye (propidium iodide, P4034, US EVERBRIGHT, Suzhou, China) was added to each sample (except the control group). All tubes were then wrapped in aluminum foil to protect them from light and incubated at 37°C for 10 minutes. To remove unbound dye, the samples were thoroughly washed with PBS before subsequent analysis. The samples were then washed twice with PBS and centrifuged at 10,000 rpm for 5 minutes. Sample data were processed and analyzed using a Cytoflex flow cytometer (BD FACSAria III, USA), recording 10,000 events.

[0036] The proportion of PI-positive cells after incubation, detected by flow cytometry, reflected the degree of cell membrane integrity impairment caused by exposure to different concentrations of TroLEAP2-21 (with PBS as a control). Gram-positive bacteria ( L. garvieae , S. epidermidis ) and Gram-negative bacteria ( V. alginolyticus , V. harveyi After being treated with 0, 3.75, 7.5, and 15 μM TroLEAP2-21 for 2 hours, L. garvieae The proportions of PI-positive cells were 1.32%, 1.66%, 1.87%, and 12.60%, respectively. S. epidermidis The percentages were 9.84%, 46%, 51.60%, and 83.10%; while V. alginolyticus The percentages were 11.80%, 13.50%, 13.40%, and 22.80%. V. harveyi The percentages were 3.73%, 32.90%, 35.20%, and 38.90%. Figure 5 Data show that changes in bacterial cell membrane permeability are positively correlated with TroLEAP2-21 concentration, and higher doses of the peptide lead to more severe membrane damage.

[0037] 4. TroLEAP2-21-induced membrane depolarization: 3,3′-dipropylthiodiacarbanium iodide (DiSC3(5)(MedChemExpress, Monmouth Junction, NJ, USA)) was used as a fluorescent probe of the cytoplasmic membrane to assess the effect of peptides on bacterial membrane potential depolarization. The method of Kwon et al. (JY Kwon, MK Kim, L. Mereuta, CH Seo, T. Luchian, Y. Park, Mechanism of action of antimicrobial peptideP5 truncations against Pseudomonas aeruginosa and Staphylococcus aureus, AMBExpress 9 (2019) 1-15.) was followed with slight modifications. Three different buffers need to be prepared for this experiment: Buffer A (Heps 0.12 g, glucose 10.4 g + 100 ml H2O), Buffer B (KCl 0.46 g + 60 ml Buffer A), and Buffer C (EDTA 120 µl + 30 ml Buffer B). Then, in a black 96-well plate, 100 μL of diluted bacterial solution (OD) 600 = 0.05) was mixed with 50 μL of 1 μM DiSC3(5) dye and incubated at room temperature for 30 minutes. Then, 50 μL of TroLEAP2-21 was added to each well (to make the final concentrations 0, 15 μM, 30 μM, and 60 μM, respectively), and the 96-well plate was placed in a microplate reader. The excitation wavelength was set to 622 nm and the emission wavelength to 670 nm. The fluorescence intensity was measured at 1-minute intervals over 30 minutes.

[0038] DiSC3(5) is a membrane potential-sensitive dye widely used to monitor changes in bacterial membrane potential, especially depolarization events. Experiments show that TroLEAP2-21 can induce depolarization in four types of bacteria (Gram-positive bacteria). L. garvieae , S. epidermidis and Gram-negative bacteria V. alginolyticus , V. harveyi The membrane depolarization was observed. Adding different concentrations of TroLEAP2-21 to a 96-well plate significantly increased the fluorescence intensity. Figure 6 It is worth noting that Gram-negative bacteria ( V. Harvey , V. alginolyticus ) and Gram-positive bacteria ( L. garvieae , S. epidermidisAll results were positive, and the higher the peptide concentration, the stronger the effect.

[0039] 5. Degradation of bacterial genomic DNA by TroLEAP2-21: This experiment was conducted according to the method of Zhang et al. (H. Zhang, Z. Cao, Q. Diao, Y. Zhou, J. Ao, C. Liu, Y. Sun, Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin, Fish & Shellfish Immunology 126(2022) 357-369.). Bacterial genomic deoxyribonucleic acid (DNA) was extracted according to the instructions of the Tiangen Bacterial Genomic DNA Extraction Kit, and the concentration and quality of the extracted DNA were detected using an Implen Nano-Photometer. 400 ng of bacterial genomic DNA was incubated with TroLEAP2-21 (final concentrations of 3.75 μM, 7.5 μM, 15 μM, 30 μM, 60 μM, and 75 μM). A positive control group treated with the same volume of DNase I and a negative control group treated with the corresponding volume of PBS were also included. Samples were incubated at room temperature for 30 minutes. The samples were then analyzed using a 1.2% agarose gel and a gel imaging system.

[0040] The ability of TroLEAP2-21 peptide to penetrate the cell membrane, bind to bacterial genomic DNA, and cause damage was detected using a gel retardation assay. Different concentration groups and positive / negative controls were set up for Gram-positive and Gram-negative bacteria. The results showed that... L. garvieae and S. epidermidis Positive results were observed at peptide concentrations of 60 μM and 75 μM. V. harveyi Significant effects were observed at 30μM, 60μM, and 75μM, while V. alginolyticus No positive results were observed. Figure 7 ).

[0041] 6. Bacterial morphological changes induced by TroLEAP2-21: Experimental design referenced (Y. Chen, L. Yao, Y. Wang, X. Ji, Z. Gao, S. Zhang, G. Ji, Identification of ribosomal protein).

[0042] L30 is an uncharacterized antimicrobial protein. (Developmental Comparative Immunology 120 (2021) 104067.) An earlier method was performed. The effects of TroLEAP2-21 on bacterial cell morphology changes were investigated using transmission electron microscopy (TEM). After washing with 0.9% NaCl, the bacterial suspension (1×10⁻⁶) was... 9 The bacterial cells (CFU / mL) were incubated with TroLEAP2-21 (final peptide concentration 30 μM) at room temperature for 1 hour. The samples were then stored in 100 mM PBS containing 2.5% glutaraldehyde, added to a 400-mesh carbon-coated grid, and incubated at room temperature for 3 minutes. Subsequently, the grid was immersed in 2% phosphotungstic acid for 3 minutes, blotted dry with filter paper, and finally the morphology and structure of the bacterial cells were observed using a JEOL JSM-840 transmission electron microscope.

[0043] The bactericidal effect of TroLEAP2-21 on bacterial cells was evaluated using transmission electron microscopy (TEM). Control group bacteria treated with 0.9% NaCl maintained their intact morphology and smooth, transparent cell membranes. In contrast, bacterial cells treated with TroLEAP2-21 showed significant morphological abnormalities and structural damage. L. garvieae and S. epidermidis Cells collapsed and their contents leaked out. V. alginolyticus and V. harveyi The cells are significantly shrunken and damaged. Figure 8 ).

[0044] 7. The role of TroLEAP2-21 in maintaining the integrity of fish tissue structure: Pathological analysis was performed on gill, liver, and intestinal tissues of fish infected with Lactococcus gasseri and Vibrio harveyi, after treatment with phosphate-buffered saline (PBS) or TroLEAP2-21 peptide. Figure 9 Infected fish treated with PBS exhibited severe gill damage, including necrosis of primary and secondary gill lamellae; however, these pathological changes were significantly alleviated in the TroLEAP2-21 treatment group, demonstrating the protective effect of the peptide. Liver tissue in the PBS control group showed lesions such as sinus varicose veins, acute cell swelling, and fatty degeneration; these injuries were significantly alleviated after TroLEAP2-21 injection. Intestinal tissue showed reduced villus height and width after PBS injection, while these parameters were significantly improved in the peptide treatment group, indicating that TroLEAP2-21 not only maintains the integrity of the intestinal epithelial structure but also promotes the recovery of nutrient absorption function.

[0045] 8. Effect of TroLEAP2-21 on individual survival rate after bacterial infection: The experiment was conducted based on the reference W.Ali, Y. Chen, Z. Wang, M. Sun, Y. Song, X. Guo, X. Wang, Y. He, J. Qi, Evaluating the

[0046] Antimicrobial Efficacy of TroLEAP2-27 peptide in golden pompano (Trachinotus ovatus) against Bacterial Pathogens, Fish & Shellfish Immunology 162 (2025) 110310. H. Zhang, Z. Cao, Q. Diao, Y. Zhou, J. Ao, C. Liu, Y. Sun, Antimicrobial activity and mechanisms of a derived antimicrobial peptide TroNKL-27 from golden pompano (Trachinotus ovatus) NK-lysin, Fish & Shellfish Immunology 126 (2022) 357-369), evaluated the in vivo efficacy of TroLEAP2-21 against bacterial infection. Before the experiment, approximately 10% of the oval pompano (Trachinotus ovatus) were randomly selected from each group. T. ovatus The presence of bacterial infection was confirmed by examination of the gills, liver, and intestinal tissues; no infection was detected. Following bacterial challenge, mortality was monitored daily for 7 days in both the TroLEAP2-21 treatment group and the PBS control group, and mortality rates were recorded. Kaplan-Meier survival curves were used to compare the survival rates of each group against *Lactococcus gasseri* (Lactococcus gasseri). L. garvieae ) or Vibrio harveyi ( V. harveyi Survival rate after infection.

[0047] By comparing the survival rates of the TroLEAP2-21 and PBS-treated groups, it was found that ( Figure 10The peptide-treated group showed significantly higher survival rates after infection with both Gram-positive bacteria (L. garvieae) and Gram-negative bacteria (V. harveyi). Furthermore, TroLEAP2-21 treatment alleviated clinical symptoms such as infection stress and loss of appetite, and histopathological studies showed less tissue damage in the peptide-treated group, confirming its important role in maintaining organ integrity. These results highlight the potential application of TroLEAP2-21 in the control of bacterial diseases in aquaculture.

[0048] 9. Cytotoxicity Assay: This experiment aimed to evaluate the cytotoxicity of TroLEAP2-21 to mammalian cells (specifically HEK 293T cells). Cell viability was assessed using the MTT assay. The principle of the MTT assay is that living cells take up yellow MTT, which is then reduced by intracellular enzymes to form blue-purple formazan crystals, which accumulate within the cell. Dead cells cannot reduce MTT and therefore do not form blue-purple crystals. The formed crystals were dissolved using the organic solvent DMSO. The absorbance of the resulting solution was then measured at a wavelength of 492 nm using a spectrophotometer. This absorbance value is correlated with the number of living cells in the sample, thus reflecting cell viability. The absorbance value of the blue-purple crystals is directly proportional to the number of living cells in the sample. The cytotoxicity assay showed that different concentrations of TroLEAP2-21 tested did not significantly affect the survival rate of HEK 293T cells (Table 1). These results preliminarily demonstrate that this peptide does not have cytotoxic effects on mammalian cells, indicating its potential safety for therapeutic applications in mammalian systems.

[0049] Table 1. Effect of antimicrobial peptide TroLEAP2-21 treatment on the survival rate of HEK 293T cells.

[0050] .

[0051] 10. Hemolysis test

[0052] Hemolysis assays were performed to assess the hemolytic activity of TroLEAP2-21. Fresh blood (5 mL) was collected from healthy oval pomfret in anticoagulant tubes and centrifuged at 1000×g for 10 min at room temperature to obtain red blood cells (RBCs). The RBCs were washed three times with lysis buffer (pH 8.2) and PBS (pH 7.4) to remove residual plasma and other components, and then resuspended in lysis buffer or PBS to a 4% (v / v) concentration. For each treatment, 200 μL of the RBC suspension was mixed with 200 μL of TroLEAP2-21 peptide solution at different final concentrations (15, 30, 60, or 120 μM). PBS and 50 μg / mL BSA were used as negative controls, and 0.1% Triton X-100 as a positive control. After incubation at 37°C for 1 hour, the samples were centrifuged at 1000×g for 10 min, and 200 μL of the supernatant was transferred to a 96-well plate. The absorbance was measured at a wavelength of 540 nm using an ELISA reader to quantify the degree of hemolysis; the higher the absorbance, the greater the degree of red blood cell lysis.

[0053] The significance of detecting whether antimicrobial peptides induce hemolysis lies in assessing their safety to host cells, particularly their effect on erythrocytes. Therefore, this experiment evaluated the hemolytic effect of TroLEAP2-21 on golden pomfret hemolymph cells. The results showed that the 0.1% Triton X-100 group exhibited significant hemolysis, while the blank control group, negative control group, and peptide-treated group showed no signs of hemolysis. Figure 11 This indicates that TroLEAP2-21 does not induce hemolysis, confirming its safety for red blood cell integrity.

Claims

1. The antimicrobial peptide TroLEAP2-21, characterized in that, The antimicrobial peptide TroLEAP2-21 is derived from the oval pomfret. The amino acid sequence of the antimicrobial peptide TroLEAP2-21 is shown in SEQ ID NO.

1. The antimicrobial peptide TroLEAP2-21 has an amphiphilic α-helix structure, that is, one side is concentrated with positively charged hydrophilic residues, and the other side is composed of nonpolar hydrophobic residues forming a hydrophobic surface.

2. The application of the antimicrobial peptide TroLEAP2-21 according to claim 1 in the preparation of antimicrobial products, characterized in that, The bacteria mentioned are Lactococcus gasseri, Staphylococcus epidermidis, Vibrio alginolyticus, and / or Vibrio harveyi.

3. The application according to claim 2, characterized in that, The antimicrobial peptide TroLEAP2-21 can alter bacterial cell membrane permeability, induce bacterial cell membrane depolarization, degrade bacterial genomic DNA, and cause morphological changes in bacteria.

4. The application of the antimicrobial peptide TroLEAP2-21 according to claim 1 in the preparation of products that maintain the integrity of fish tissue structure, characterized in that, The application involves using the antimicrobial peptide TroLEAP2-21 to repair damage to the body tissues of oval pomfret.

Images