Immunoactive peptide RM from perinereis aibuhitensis and application thereof

Abstract

The invention discloses a perinereis aibuhitensis-sourced immunoactive peptide RM and application thereof, and belongs to the technical field of bioactive peptides. According to the invention, lt; the method comprises the following steps: by taking a 5 kDa nereis enzymatic hydrolysate as a raw material, separating and purifying through Sephadex G-25 gel chromatography to obtain a nereis purified component, infecting nematodes by utilizing pseudomonas aeruginosa PA14 to establish an immunocompromised nematode model, and evaluating the immunocompetence of the nereis purified component by measuring the life, exercise ability, reproductive ability and lipofuscin accumulation indexes of the nematodes. A peptide spectrum of a polypeptide component with optimal immunocompetence is represented by combining a polypeptiomics technology, immunocompetence peptide is rapidly screened out by combining bioinformatics and a molecular docking technology, finally, nereis active peptide is obtained through chemical synthesis, the immunocompetence is verified by utilizing nematodes infected by PA14, and a reference is provided for rapid screening of the food-borne immunocompetence peptide.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to an immunologically active peptide RM derived from *Nematocystis didentata* and its applications. Background Technology

[0002] Sandworms, also known as sea centipedes, sea leeches, and sandworms, are marine annelids that live on muddy sandy beaches in the intertidal zone. Rich in protein, amino acids, unsaturated fatty acids, trace elements, and vitamins, sandworms possess high nutritional and medicinal value. Ancient herbal texts record their use in treating various ailments, including strengthening the spleen and stomach, nourishing blood, promoting diuresis and reducing swelling, and dispelling wind and dampness. Their edible and medicinal value is widely recognized.

[0003] Studies have shown that sandworms possess anticoagulant, thrombolytic, and antitumor effects, and have a long history of application in traditional Chinese medicine. Currently, there are few reports on the detailed structure and activity of polypeptide components derived from sandworms. Summary of the Invention

[0004] The purpose of this invention is to provide an immunologically active peptide RM derived from *Pteris vittata* and its applications, in order to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is an immunologically active peptide derived from *Pteris vittata*, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] The second technical solution of the present invention is the application of the immune-active peptide in the preparation of products that enhance immunity.

[0007] The third technical solution of the present invention is a product for enhancing immunity, comprising the aforementioned immune-active peptides.

[0008] The fourth technical solution of the present invention is the application of the immune-active peptide in the preparation of products that extend lifespan.

[0009] The fifth technical solution of the present invention is a product for extending lifespan, comprising the aforementioned immune-active peptide.

[0010] The sixth technical solution of the present invention is the application of the immune-active peptide in the preparation of products that enhance reproductive capacity.

[0011] The seventh technical solution of the present invention is a product for improving reproductive capacity, comprising the aforementioned immune-active peptide.

[0012] The eighth technical solution of the present invention is the application of the immune-active peptide in the preparation of products that inhibit the accumulation of lipofuscin.

[0013] The ninth technical solution of the present invention is a product for inhibiting the accumulation of lipofuscin, comprising the aforementioned immune-active peptide.

[0014] Based on the above technical solution, the present invention has the following technical effects: This invention uses <5 kDa *Neemum sparganum* enzymatic hydrolysis products as raw materials. After purification by Sephadex G-25 gel chromatography, purified *Neemum sparganum* fractions are obtained. An immunocompromised nematode model is established by infecting *Pseudomonas aeruginosa* PA14. The immunogenicity of the purified *Neemum sparganum* fractions is evaluated by measuring nematode lifespan, motility, reproductive capacity, and lipofuscin accumulation. Peptidomics technology is used to characterize the peptide profile of the most immunogenic peptides. Furthermore, bioinformatics and molecular docking techniques are combined to rapidly screen for immunogenic peptides. Finally, *Neemum sparganum* bioactive peptides are obtained through chemical synthesis, providing a reference for the rapid screening of foodborne immunogenic peptides. Attached Figure Description

[0015] Figure 1 The image shows the gel chromatography separation and purification pattern of the <5 kDa *Nematocystis jirovecii* ultrafiltration fraction G25.

[0016] Figure 2 The effect of purified components from *Nematodes spp.* on the lifespan curves of *Nematodes spp.* (a)-P1; (b)-P2; (c)-P3; (d)-P4.

[0017] Figure 3 The effect of purified components from *Nematodes spp.* on the number of ovipositions in *Nematodes spp.* was investigated. Specifically, compared to the CT group, #... ###P< 0.001 ;*. Compared with the MD group, *P<0.05, **P<0.01 , ***P<0.001 .

[0018] Figure 4 The effect of purified components from *Nematocystis jirovecii* on lipofuscin accumulation in *Nematocystis jirovecii*. (a) - Fluorescence image of lipofuscin accumulation; (b) - Relative fluorescence intensity of lipofuscin magnified by 40x; #. Compared with the CT group, ### P<0.001 *. Compared with the MD group, *P<0.05, **P<0.01, ***P<0.001 .

[0019] Figure 5 Biological information analysis of a candidate peptide library of active peptides from *Nematocystis jirovecii*. (a) - terminal amino acid characteristics; (b) - isoelectric point; (c) - instability coefficient; (d) - aliphatic coefficient; (e) - average hydrophilicity coefficient.

[0020] Figure 6The diagram shows the 3D structure and interaction between the active peptide from *Nereis spp.* and the TLR2 molecule. (a) shows the molecular interaction between RM and TLR2 (TLR2 (PDB ID: 6NIG)); (b) shows a 2D image of the interaction site between RM and the receptor.

[0021] Figure 7 The diagram shows the 3D structure and interaction between the active peptide from *Nereis externa* and the TLR2 molecule. (a) shows the molecular interaction between KP and TLR2 (TLR2 (PDB ID: 6NIG)); (b) shows a 2D image of the interaction site between KP and the receptor.

[0022] Figure 8 The diagram shows the 3D structure and interaction between the active peptide from *Nereis spp.* and the TLR2 molecule. (a) shows the molecular interaction between PP and TLR2 (TLR2 (PDB ID: 6NIG)); (b) shows a 2D image of the interaction site between PP and the receptor.

[0023] Figure 9 The images show the HPLC and mass spectra of the solid-phase synthesis of nereis peptide RM. (a) is the HPLC chromatogram of RM; (b) is the mass spectrum of RM.

[0024] Figure 10 The images show the HPLC and mass spectra of the solid-phase synthesis of nereisin KP. (a) is the HPLC chromatogram of KP; (b) is the mass spectrum of KP.

[0025] Figure 11 The images show the HPLC and mass spectra of nereistocin (PP) synthesized in the solid phase. (a) is the HPLC chromatogram of PP; (b) is the mass spectrum of PP.

[0026] Figure 12 The effects of the active peptide RM from *Nematodes nigra* on the lifespan curves of nematodes were investigated. Specifically, (a) RM was used to treat wild-type N2 nematodes; (b) RM was used to treat km25 gene-deficient nematodes; and (c) RM was used to treat zu135 gene-deficient nematodes.

[0027] Figure 13 The effect of the active peptide RM from *Nematocystis jirovecii* on lipofuscin accumulation in wild-type N2 nematodes. (a) - Fluorescence image of lipofuscin accumulation; (b) - Relative fluorescence intensity of lipofuscin magnified by 40x; #. Compared with the CT group, ### P< 0.001 ;*. Compared with the MD group, *P<0.05, **P<0.01, ***P<0.001 .

[0028] Figure 14The effect of the active peptide RM from *Nematocystis jirovecii* on lipofuscin accumulation in *PMK-1(KM25)* gene-deficient nematodes. (a) - Fluorescence image of lipofuscin accumulation; (b) - Relative fluorescence intensity of lipofuscin magnified by 40x; #. Compared with the CT group, ### P<0.001 ;*. Compared with the MD group, *P<0.05, **P<0.01, ***P<0.001 .

[0029] Figure 15 The effect of active peptides from *Nematocystis jirovecii* on lipofuscin accumulation in *Skn-1(Zu135)* gene-deficient nematodes. (a) - Fluorescence image of lipofuscin accumulation; (b) - Relative fluorescence intensity of lipofuscin magnified by 40x; #. Compared with the CT group, ### P<0.001 ;*. Compared with the MD group, *P<0.05, **P<0.01, ***P<0.001 . Detailed Implementation

[0030] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0031] This invention provides an immunologically active peptide derived from *Pteris vittata*, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0032] This invention also provides the application of the immunomodulatory peptide in the preparation of products that enhance immunity.

[0033] This invention also provides a product for enhancing immunity, comprising the aforementioned immune-active peptides.

[0034] This invention also provides the application of the immunologically active peptide in the preparation of life-extending products.

[0035] This invention also provides a product for extending lifespan, comprising the aforementioned immune-active peptide.

[0036] This invention also provides the application of the immunologically active peptide in the preparation of products that enhance reproductive capacity.

[0037] This invention also provides a product for improving reproductive capacity, comprising the aforementioned immune-active peptide.

[0038] This invention also provides the application of the immunologically active peptide in the preparation of products that inhibit lipofuscin accumulation.

[0039] This invention also provides a product for inhibiting lipofuscin accumulation, comprising the aforementioned immunomodulatory peptide.

[0040] Example 1 1. Materials and Instruments 1.1 Raw materials and reagents *Nematocystis jirovecii*, purchased from Suixi Tengfei Biotechnology Co., Ltd.; N2 wild-type *C. elegans* (… Caenorhabditis elegans The following were purchased from the Center for Genetics of Caenorhabditis elegans (CGC) at the University of Minnesota: *C. elegans* bioactive peptides KP, RM, and PP (white powder, purity >95%), purchased from Shanghai Sangon Biotech Co., Ltd.; U25 [pmk-1 (km25)] IV EU31 [skn-1 (zu135)] IV Gene-deficient nematodes were purchased from the Center for Genetic Research on Caenorhabditis elegans (CGC) at the University of Minnesota; Caenorhabditis elegans were cultured at 20°C on Nematode Growth Medium (NGM); OP50 Escherichia coli (… E. coli OP50 (uracil-deficient type) was purchased from Fujian Shangyuan Biotechnology Co., Ltd.; Pseudomonas aeruginosa PA14 ( Pseudomonas aeruginosa 14) Purchased from BSBCC plasmid strain library; NGM medium, medium additives A and B solution, Shijiazhuang Ximo Technology Co., Ltd.; LB solid medium and LB liquid medium, Beijing Luqiao Biotechnology Co., Ltd.; sodium chloride, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, sodium hydroxide, Xilong Technology Co., Ltd.; 10% sodium hypochlorite, 5-fluoro-2-deoxyuridine (FUDR), Ron Biotech Co., Ltd.

[0041] 1.2 Reagent Preparation NGM medium: Dissolve 22.6 g of NGM medium powder in 1 L of distilled water, heat in a water bath until completely dissolved, then autoclave at 121°C for 15 min. After cooling to 50-60°C, add 10 culture medium additives, shake well, and then pour into plates for later use.

[0042] LB broth: Dissolve 25.0 g LB broth in 1 L of pure water, autoclave at 121°C for 15 min, inoculate with E. coli and store under cold for later use.

[0043] M9 buffer: 2.5 g sodium chloride, 3 g sodium dihydrogen phosphate, 0.125 g magnesium sulfate, and 1.5 g potassium dihydrogen phosphate are brought to a final volume of 500 ml. The solution is then autoclaved at 121°C for 15 min before use.

[0044] Nematode lysate: 1 mol / L sodium hydroxide: 10% sodium hypochlorite: sterile water = 1:1:8 ratio, store at 4℃ protected from light, prepare and use immediately.

[0045] 1.3 Instruments and Equipment Table 1 Instruments and Equipment

[0046] 2 Experimental Methods 2.1 Ultrafiltration Separation and Purification of Components from *Nematocystis jirovecii* Based on the immunomodulatory effect of *Nereis bisporus* enzymatic hydrolysate on immunosuppressed mice (Journal of Ocean University of Taiwan, 2025, 40(1): 76-85), *Nereis bisporus* ultrafiltration fractions with a concentration of <5 kDa were obtained and separated using a Sephadex G-25 gel in an AKTA-Puifier protein purification instrument. Separation conditions: sample concentration 10 mg / mL, flow rate 5.0 mL / min, eluent was ultrapure water (Φ=0.22 μm), detection wavelength 280 nm. Four purified *Nereis bisporus* fractions, P1, P2, P3, and P4, were obtained. These fractions were concentrated by rotary evaporation and lyophilized to obtain lyophilized powders of the four purified *Nereis bisporus* fractions.

[0047] 2.2 Cultivation and Synchronization Treatment of Nematodes Coated with E. coli OP50 bacterial culture (OD) 600 Nematode growth culture with a ratio of 0.4~0.6 was conducted in a 20℃ constant temperature incubator. The nematodes were synchronized using the NaClO lysis method. After discarding the supernatant, the nematode eggs obtained from the lysis were collected and added to NGM plates. After culturing in a 20℃ constant temperature incubator for 48 h, they developed into L4 stage larvae, completing the synchronization treatment.

[0048] 2.3 Activity Evaluation Experiment Grouping of Purified Components from Polygonatum spp. Nematodes synchronized to the L4 stage were collected and set up as a blank control group (CT), a Pseudomonas aeruginosa PA14 (hereinafter referred to as PA14) infection model group (MD), and four treatment groups with different mass concentrations of purified nematodes (P1, P2, P3, P4) at 20 μg / mL, 50 μg / mL, and 100 μg / mL, with three replicates in each group.

[0049] 2.4 PA14 Infection of N2 Wild-Type Nematodes Lifespan Test PA14 was streaked on LB plates and incubated at 37°C for 24 h. Single colonies were picked and incubated in LB broth at 37°C and 180 rpm. The OD of PA14 was then measured. 600 =0.10±0.05), take 20, 50 and 100 μL of bacterial culture respectively and spread it onto NGM plates with a mass concentration of 10 μg / mL 5-fluoro-2-deoxyuridine (FUDR), incubate at 37℃ for 12 h, transfer to 25℃ for 12 h, and that is the PA14 infection plate.

[0050] Thirty L4-stage N2 nematodes were selected and spread onto NGM medium containing OP50 Escherichia coli to establish a CT group and a PA14 model group. They were naturally cultured at 20°C, and this was recorded as day 0. After 4 days, nematodes from the PA14 model group were transferred to prepared PA14 infection plates (freshly prepared and used immediately) and incubated at 25°C for 24 hours. The surviving nematodes were then transferred to fresh NGM medium. The CT group received only OP50 bacterial suspension daily and no other treatment. The number of nematode deaths and survivors was counted daily until all nematodes died.

[0051] 2.5 Lifespan and number of head-tail swings of wild-type N2 nematodes L4 nematodes were cultured simultaneously using different mass concentrations (20, 50, and 100 μg / mL) of four polychaete purification components (P1, P2, P3, and P4). Each group had three replicates with 30 nematodes per replicate, and the cultures were incubated at 20°C. Ultrapure water was used instead of the polychaete purification components in the CT and MD groups.

[0052] From the date of transfer as day 0, L4 nematodes were simultaneously cultured at 20, 50, and 100 μg / mL of the four purified polychaete fractions for 4 days. Then, together with the MD group, the nematodes were picked into PA14 infection plates and incubated at 25℃ for 24 h. The surviving nematodes were then transferred to new NGM medium. The number of dead and surviving nematodes was counted daily until all individuals died. Survival curves were plotted based on the survival and death time of the nematodes.

[0053] After N2 nematode infection, observe and record the nematode's motility on days 7, 9, and 10. The specific procedure is as follows: Add an appropriate amount of M9 buffer to the center of a sterile culture dish, transfer the nematode to be tested into the droplet, and after acclimatizing for 20 seconds, record the number of whole-body swings completed within 10 seconds (judgment criteria: one complete reverse swing of the nematode's head and tail and its return to its original position is counted as one swing).

[0054] 2.6 Reproductive capacity test of N2 wild-type nematodes L4 nematodes were cultured simultaneously using different mass concentrations (20, 50, and 100 μg / mL) of four polychaete purification components (P1, P2, P3, and P4). Each group had five replicates, with one nematode per replicate, and the cultures were incubated at 20°C. Ultrapure water was used instead of the polychaete purification components in the CT and MD groups.

[0055] From day 0 of the transfer, L4 nematodes were simultaneously cultured in NGM culture plates (without FUDR) at concentrations of 20, 50, and 100 μg / mL using four different purified nematode components. After 2 days, the nematodes were transferred to PA14 infection plates (without FUDR) together with the MD group. After 24 hours of incubation at 25°C, the surviving nematodes were transferred to new NGM medium, while the old NGM and PA14 infection plates were kept and cultured at 20°C. The number of nematodes in the old NGM culture plates was counted daily. The number of nematode eggs and larvae was counted daily, and the plates were transferred once a day. The count of nematode eggs and larvae was repeated until the adults stopped laying eggs. The total number of eggs and larvae was then counted.

[0056] 2.7 Lipofuscin Accumulation Test of N2 Wild-Type Nematodes L4 nematodes were cultured simultaneously using different mass concentrations (20, 50, and 100 μg / mL) of four polychaete purification components (P1, P2, P3, and P4), with three replicates in each group. The cultures were incubated in a 20°C constant temperature incubator. Ultrapure water was used instead of polychaete purification components in the CT and MD groups.

[0057] From day 0 of transfer, L4 nematodes were simultaneously cultured at concentrations of 20, 50, and 100 μg / mL using four different purified polychaete fractions, respectively, for 4 days. Then, together with the MD group, the nematodes were selected and placed into PA14 infection plates. After 24 hours of incubation at 25°C, surviving nematodes were transferred to slides coated with 2% agarose and anesthetized with 1 g / L tetraimidazole hydrochloride solution. Observation and image acquisition were performed using a fluorescence microscope. ImageJ software was used to statistically analyze the fluorescence intensity to determine the relative content of lipofuscin in the nematodes.

[0058] 2.8 Identification of polypeptide sequences of purified fibroids from *Nematocystis jirovecii* We commissioned Biotech to perform peptide sequence identification on the purified nervone fragments and used liquid chromatography-mass spectrometry (LC-MS / MS) to identify the nervone fragments with the strongest immunogenicity.

[0059] First, the sample was dissolved, reductively alkylated, eluted using a C18 column, and vacuum dried. Peptides were then quantified. Samples were analyzed using a NanoViper C18 (75 μm × 25 cm, 1.9 μm) column. Liquid conditions were as follows: eluent A consisted of 0.1% formic acid and deionized water; eluent B consisted of 0.1% formic acid and 80% ACN; flow rate was 600 nL / min; and analysis time for each fraction was 66 min. The elution program was as follows: 0–2 min, 4% B; 2–35 min, 8% B; 35–55 min, 28% B; 55–56 min, 40% B; 56–66 min, 95% B. The full scan range of mass spectrometry was 100-1500 m / z. The resolution of the first-stage mass spectrometry was set to 120,000, the AGC was set to Standard, and the Maximum IT was set to 20 ms. High-energy collisional fragmentation (HCD) was used for fragmentation, followed by second-stage mass spectrometry detection. The resolution of the second-stage mass spectrometry was set to 15,000, the AGC was set to Standard, the Maximum IT was set to 22 ms, and the peptide fragmentation collision energy was set to 30.

[0060] 2.9 Screening of Potential Immunoreactive Peptides PeptideRanker was used to predict the potential biological activity of the identified peptide sequences. A prediction score threshold of 0.7 was set to further evaluate the peptide sequences for predicting potential biological activity. The BIOPEP database was used to verify whether the immunological activity of the peptide sequences had been validated. Expasy ProtoParam was used to analyze the physicochemical properties of the screened peptides, such as molecular weight (WM), isoelectric point (pI), and mean hydrophilicity (GRAVY). In the functional food field, detecting the toxicity of peptides is also very important; the ToxinPred tool was used to predict the potential toxicity of peptide sequences.

[0061] 2.10 Molecular docking The crystal structures of TLR2 (PDB ID: 6NIG) and TLR4 / MD-2 (PDB ID: 5IJD) were obtained from the RCSB PDB database. Small peptide ligands were plotted using ChemDraw and Chem3D 22.0.0 software. The peptides were docked with TLR2 and TLR4 / MD-2 using AutoDockTools 1.5.6 software, and the binding ability of the docking systems was scored. The interaction between the protein receptor and peptide ligand was visualized using Pymol software. The interaction forces between the protein receptor and peptide ligand were processed using Discovery Studio 2019 Client software.

[0062] 2.11 Chemical Synthesis of Nephrolepis externa Active Peptides The active peptides of *Nematocystis jirovecii* were synthesized by Sangon Biotech Co., Ltd. using a solid-phase synthesis method. The purity of the synthesized peptides was detected and identified using high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS).

[0063] 2.12 Experimental grouping for evaluating the activity of *Nephropus neriifolius* bioactive peptides Three different concentrations of three active peptides from *Nematocystis aeruginosa*—a blank control group (CT), a *Pseudomonas aeruginosa* PA14 infection model group (MD), and treatment groups—RPPLYGSM (RM), KFLNPPP (KP), and PPGMRGPPP (PP)—were set up, with 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, respectively. Each group had three replicates.

[0064] 2.13 Lifetime assay of PA14 infected nematodes with defects in pmk-1 (km25) and skn-1 (zu135) genes PA14 was streaked on LB plates and incubated at 37°C for 24 h. Single colonies were picked and incubated in LB liquid at 37°C and 180 rpm. The OD600 of PA14 was measured (0.07±0.01). 5, 10, 15, 20, and 50 μL of bacterial suspension were spread onto NGM plates containing 10 μg / mL 5-fluoro-2-deoxyuridine (FUDR, to prevent progeny interference with the assay) to prepare PA14 infection plates.

[0065] Thirty L4-stage km25 and zu135 gene-deficient nematodes were selected and cultured on NGM medium coated with OP50 Escherichia coli to establish a CT group and a PA14 model group. The nematodes were incubated naturally at 20°C, and this was recorded as day 0. After 4 days, the nematodes from the PA14 model group were transferred to freshly prepared PA14 infection plates and incubated at 25°C for 24 hours. The surviving nematodes were then transferred to fresh NGM medium. The blank control group received only OP50 bacterial suspension daily and received no other treatment. The number of nematode deaths and survivors was counted daily until all nematodes died.

[0066] 2.14 Lifetime test of N2 wild-type and gene-deficient nematodes The method is the same as in 2.4 for life determination.

[0067] 2.15 Lipofuscin Accumulation Assay in N2 Wild-type and Gene-Deficient Nematodes The method is the same as in 2.7.

[0068] 2.16 Data Statistics and Analysis The data were analyzed using IBM SPSS Statistics 27 software with one-way ANOVA and Tukey post-hoc test to determine differences between groups. The results are expressed as mean ± standard deviation. P<0.05 The differences were considered statistically significant; graphs were plotted using GraphPad Prism 6 and Origin 2021.

[0069] 3 Results and Analysis 3.1 Activity evaluation of purified components from *Nereidum spp.* 3.1.1 Results of G25 gel chromatography separation and purification of <5 kDa *Nematocystis jirovecii* ultrafiltration fraction Four purified fractions of *Nereidum spp.* were obtained by G25 gel chromatography separation and purification of the <5 kDa ultrafiltration fraction, namely P1, P2, P3, and P4 (see...). Figure 1 ).

[0070] 3.1.2 Purified components from *Nematodes nervosa* prolong the lifespan of PA14-infected nematodes. The mean lifespan of nematodes in the CT group was 12.22 ± 0.04 days, while the mean lifespan of nematodes in the PA14-infected MD group was 8.34 ± 0.15 days, showing a significant difference compared to the CT group. P<0.05 The average lifespan was shortened by 31.73%, and the longest lifespan was shortened by 6 days, indicating that PA14 infection of nematodes leads to weakened immunity in nematodes, ultimately resulting in a shortened lifespan (Table 2).

[0071] Table 2. Effects of purified components from *Nematodes* on nematode lifespan.

[0072] Note: Different letters in each column indicate that there is a significant difference between the two groups. P<0.05 ).

[0073] After adding the purified components P1, P2, P3, and P4 from the nematode, the average and maximum lifespan of the nematodes were extended to varying degrees, and their lifespan curves shifted to the left compared to the MD group. Figure 2We also found that, except for P2, the average lifespan of nematodes treated with P1, P3, and P4 increased by 1.87%–15.05% with increasing concentration, indicating that the purified components of *Nematodes nereis* (P1, P3, and P4) have a dose-dependent effect on nematode lifespan. At low concentrations, the effect may be weaker, resulting in a weaker ability to restore nematode immune damage. However, at higher concentrations, they exhibit optimal biological activity, approaching that of the CT group. Specifically, intervention with 100 μg / mL P1 in immunocompromised nematodes extended their average lifespan and maximum lifespan by 15.05% and 3 days, respectively, with the longest lifespan extension. These results indicate that the purified components of *Nematodes nereis* can enhance the immunity of PA14-infected nematodes to varying degrees, restore nematode immune damage, and prolong nematode lifespan.

[0074] 3.1.3 Purified components of *Nematodes nervosa* improve the motility of PA14-infected nematodes. Nematode movement behavior can reflect changes in the function of motor neurons. The nervous system senses external stimuli through neurons, regulates innate immune stress responses, and maintains cellular homeostasis. The number of body swings in nematodes reflects their movement behavior and physiological health status. Therefore, changes in immune stress in nematodes can be reflected by the number of body swings. In this invention, after nematodes were infected with PA14, the number of body swings decreased continuously with the extension of culture time. On days 7, 9, and 10, the number of body swings in the MD group was significantly reduced compared to the CT group (P<0.001) (Table 3), indicating that the model was successfully established.

[0075] Nematodes treated with purified polychaete fractions P1, P2, P3, and P4 showed increased body wobble frequency and enhanced motor ability at days 7, 9, and 10 compared to the MD group. This indicates that the purified polychaete fractions have a protective effect on the nervous system of nematodes infected with PA14, enhancing their motor ability by improving their immunity and strengthening the connections between neurons. At day 7, the number of body wobble frequency in P1-treated nematodes was significantly increased compared to the MD group (P<0.01); at day 9, except for P2 and 20 μg / mL P1, the number of body wobble frequency in nematodes treated with the other purified polychaete fractions was significantly increased (P<0.05); at day 10, the number of body wobble frequency in nematodes treated with P1 and 100 μg / mL P3 was significantly increased compared to the MD group (P<0.01). In addition, the intervention of 100 μg / mL P1 in nematodes increased the number of body swings by 40.50%, 68.62%, and 79.38% on days 7, 9, and 10, respectively, compared with the MD group (Table 3). It had the best effect on slowing down the decline in the motility of PA14-infected nematodes, prolonging the lifespan of nematodes and improving their overall health.

[0076] Table 3. Effects of purified polychaete components on the number of body swings in nematodes.

[0077] Note: #. Compared with the CT group, ### P<0.001 ;*. Compared with the MD group, *P<0.05, **P<0.01, ***P< 0.001 .

[0078] 3.1.4 Purified components of *Nematodes nigra* protect the reproductive capacity of PA14-infected nematodes. The reproductive capacity of nematodes is one of the fundamental criteria for defining life, and the number of eggs laid reflects their reproductive capacity. The relationship between reproductive capacity and the immune system is a complex and multifaceted process, involving a trade-off. According to the principle of energy conservation, when an organism is stressed by exogenous factors, it often enhances its immune defenses, weakens its reproductive capacity, and increases its survival rate. When nematodes are infected by pathogens, the immune response is activated, consuming a large amount of energy and resources, while simultaneously inhibiting their reproductive capacity. Furthermore, toxins produced by pathogens can directly damage the reproductive cells of nematodes. After infection with PA14, the virulence factors and effector proteins produced by nematodes catalyze the elongation factor of the nematode reproductive system proteins through intracellular and extracellular transport, causing the elongation factor to undergo glycosylation and lose its activity, preventing intracellular protein synthesis, leading to reproductive cell death, damaging the nematode reproductive system, weakening its reproductive capacity, and shortening its reproductive cycle. Therefore, this invention evaluates the immunomodulatory effects of purified components from *Nematodea spp.* by assessing the egg-laying rate of immunocompromised nematodes induced by PA14.

[0079] Compared with the CT group, the number of ovipositions of nematodes was significantly reduced in the MD group. P<0.001 This indicates that PA14 infection weakens the reproductive capacity of nematodes; compared with the MD group, the number of eggs laid by nematodes treated with P1, P2, P3, and P4 all increased, with P1 and P3 showing a dose-dependent effect on the number of eggs laid; while there was no significant difference in the P2 treatment group. P>0.05 P4 significantly increased the number of ovipositions in nematodes at a concentration of 100 μg / mL. P<0. 05) Figure 3 The results showed that the purified components of *Nematodes nervosa* had a protective effect on nematodes whose reproductive capacity was weakened after infection with PA14, and the 100 μg / mL treatment group of P1 had the strongest effect on protecting the oviposition capacity of nematodes.

[0080] 3.1.5 The purified components of *Nematodes nervosa* reduced the accumulation of lipofuscin in PA14-infected nematodes. The innate immune system of nematodes is closely related to free radicals. During the immune response, activated immune cells such as neutrophils and macrophages produce a large number of free radicals to fight pathogens. However, excessive free radical production can lead to tissue damage, lipid oxidation, and lysosomal degradation, resulting in an increase in lysosomal digestion products and lipid residues. This, in turn, leads to the accumulation and precipitation of lipofuscin in the nematode, producing autofluorescence. Therefore, the higher the free radical content, the higher the lipofuscin content, and the deeper the fluorescence color. Lipofuscin accumulation can also cause inflammatory responses, damage immune cells, weaken the immune system, reduce the body's immune response capacity, cause slow death, and shorten the nematode's survival time, reducing its average lifespan.

[0081] To further investigate the effects of purified polychaete components on PA14-induced immunosuppressive nematodes, this invention elucidates the immunomodulatory effects of purified polychaete components on the innate immune function of nematodes by measuring lipofuscin levels, which reflects changes in free radicals in nematodes after PA14 infection.

[0082] The results are as follows Figure 4 As shown, compared with the CT group, the accumulation of lipofuscin in nematodes in the MD group was significantly increased. P< 0.001 Compared with the MD group, nematodes treated with purified polysaccharide components P1, P2, P3, and P4 all showed reduced accumulation of lipofuscin in their bodies, with the accumulation decreasing gradually with increasing concentration, exhibiting a concentration-dependent effect. These results indicate that the purified polysaccharide components can enhance the immunity of nematodes infected with PA14 to varying degrees, reduce immune damage, and improve nematode health by inhibiting the generation of free radicals and reducing lipofuscin accumulation. The 100 μg / mL P1 treatment showed the most significant effect in reducing lipofuscin accumulation in nematodes.

[0083] This invention investigates the immunomodulatory effects of purified polychaete components P1, P2, P3, and P4 on PA14-infected nematodes in terms of lifespan, motility, reproductive capacity, and lipofuscin accumulation in N2 wild-type nematodes. The results show that the purified polychaete components can enhance the immunity of PA14-infected nematodes, reduce immune damage, prolong lifespan, slow the decline in motility, protect oviposition capacity, reduce lipofuscin accumulation, and improve overall health. Among these, intervention with 100 μg / mL P1 showed superior immunomodulatory effects compared to the other three components.

[0084] 3.2 Identification of purified polypeptide components from *Nematocystis jirovecii* and establishment of a candidate peptide library 3.2.1 Bioinformatics Feature Analysis of Candidate Peptide Libraries Studies have shown that the immunomodulatory activity of peptides obtained from protease hydrolysates is closely related to the peptide's length, sequence, amino acid composition, hydrophobicity, charge, and structure. To investigate whether the P1 component possesses the characteristics of immunomodulatory peptides, further peptide sequence identification and screening for immunomodulatory peptides are necessary.

[0085] P1 peptides were identified by LC-MS / MS, yielding 1593 peptides with 7–29 amino acids and molecular weights ranging from 563 to 2919 Da. Potential biological activity was predicted based on the identified peptide sequences. A prediction score >0.7 was considered indicative of potential biological activity. Studies showed that peptide sequences with potential immunomodulatory activity are characterized by low molecular weight (2–10 amino acid residues) and hydrophobicity. Based on these criteria, 135 peptide sequences were screened, resulting in a final selection. Physicochemical properties of the selected peptide sequences were analyzed, including molecular weight, isoelectric point, average hydrophilicity coefficient, instability coefficient, and aliphatic coefficient, to establish a candidate peptide library.

[0086] The amino acid arrangement characteristics of the candidate peptide library of this invention are as follows: the first amino acid at the C-terminus is mainly composed of proline (P), phenylalanine (F), leucine (L), and cysteine ​​(C), of which three are hydrophobic amino acids; the C2 and C3 ends are mainly occupied by proline (P), phenylalanine (F), and leucine (L), all of which are hydrophobic amino acids. Figure 5 (a) Studies have shown that most immunomodulatory peptides contain specific amino acid compositions at the N- or C-terminus of amino acids, such as hydrophobic amino acids (valine, leucine, proline, tryptophan, alanine, isoleucine, methionine, and phenylalanine) and aromatic amino acids (tyrosine, tryptophan, and phenylalanine). Furthermore, branched-chain amino acids (leucine, isoleucine, and valine) have been shown to activate the m-TOR signaling pathway, which is involved in enhancing innate and adaptive immune responses, and the hydrophobic structure of branched-chain amino acids also confirms their immunomodulatory peptide properties.

[0087] The molecular weights of the 135 candidate peptide sequences in the peptide library are concentrated between 700-1000 Da, accounting for 88.89%, and the isoelectric points of the peptide library are mostly concentrated between 5 and 9, accounting for 78.52%. Figure 5 In (b), 52 peptide sequences in the peptide library have an instability coefficient below 60. Figure 5 In (c), the aliphatic coefficient refers to the relative volume occupied by aliphatic side chains (phenylalanine, valine, isoleucine, and leucine). In the peptide library, 103 peptides have an aliphatic coefficient greater than 39, indicating the presence of a large number of aliphatic side chains. Figure 5 In the middle (d)), the hydrophilicity coefficient is usually between -2 and 2. The larger the value, the stronger the hydrophobicity. There are 45 peptide sequences in the peptide library with a hydrophilicity coefficient > 0. Figure 5(e) Scientific research has found that immunologically active peptides often contain hydrophobic amino acid residues, such as Phe, Leu, Pro, and Gly. Furthermore, positively charged peptides exhibit stronger chemotaxis and bind more strongly to receptors on the surface of immune cells, activating immune signaling pathways and stimulating immune responses. Other studies have shown that the high isoelectric point of peptides promotes immune cell function and cytokine secretion. Therefore, the various physicochemical properties of candidate peptide libraries, including characteristic amino acids, high hydrophobicity, and high isoelectric point, may be beneficial for screening immunologically active peptides.

[0088] 3.2.2 Screening and Molecular Docking of Immune Active Peptides from Nereidum Fifteen peptide sequences were screened based on characteristic amino acids and high isoelectric point, and molecularly docked with the binding pockets of Toll-like receptors TLR2 (PDB ID: 6NIG) and TLR4 / MD-2 (PDB ID: 5IJD). The interaction mechanism between the peptides and receptor proteins was analyzed. Toll-like receptors are cell membrane receptors; immunomodulatory peptides bind to them, activating signaling cascades in immune cells, promoting transcriptional activation and cytokine production. Once activated, TLRs act on the body's innate immunity and are widely present in various immune system cells, such as macrophages, granulocytes, mast cells, and dendritic cells. They can recognize various microbial antigens or extracellular stimuli and are widely considered upstream membrane recognition receptors of innate immune cells. Studies have shown that TLR2 and TLR4 can recognize and trigger downstream signaling pathways, including the MAPK and NF-κB pathways involved in adaptive immune gene expression. They activate cytoplasmic transcription factors by recognizing pathogen-associated molecular patterns, thereby activating the body's immune response.

[0089] In summary, this invention selected TLR2 (PDB ID: 6NIG) and TLR4 / MD-2 (PDB ID: 5IJD) receptors and 15 peptides for docking. Binding energy is generally considered a reference value for predicting the interaction between peptide sequences and receptor proteins; a smaller value indicates a lower binding energy for ligand-receptor interaction and a greater likelihood of forming a more stable molecular binding conformation. Generally, ligand-receptor binding energies ≤ -7 kcal / mol are considered to have strong binding activity. The docking results of this invention are shown in Table 4. 80% of the 15 screened peptides had docking energies less than -7.0 kcal / mol. Among them, SEQ ID NO.1: RPPLYGSM (named RM), SEQ ID NO.2: KFLNPPP (named KP), and SEQ ID NO.3: PPGMRGPPP (named PP) all exhibit positive charge, high isoelectric point, and a high proportion of hydrophobic amino acids. These characteristics are beneficial for the peptides to promote the function of immune cells and the secretion of cytokines, enhance the body's immune regulation function, and activate immune signaling pathways. Furthermore, from... Figure 6-8 It is evident that RM, KP, and PP can successfully dock with TLR2 and TLR4 / MD-2, forming stable complex structures through hydrogen bonds, van der Waals forces, and hydrophobic interactions. This complex structure facilitates strong binding, enabling the *Nephrolepis externa* active peptides to activate immune signaling pathways and regulate the body's immunity.

[0090] Table 4. Molecular docking results and physicochemical property analysis of active peptides from *Nematocystis jirovecii*.

[0091] 3.2.3 Solid-phase synthesis of active peptides from *Nereidum spp.* RM, KP, and PP were synthesized using a solid-phase synthesis method. The purity of the synthesized nereis terrestris active peptide was analyzed by HPLC. Figure 9-11 The specific peak times and peak areas are shown in Table 5. Peak a-4 is RM, peak b-3 is KP, and peak c-3 is PP. The content was calculated based on the peak area, and the final actual purity was all above 95%. The sequences were identified using MS / LC-MS. The mass spectra showed that the mass-to-nucleus ratio of the synthesized peptide RM was 920.00 (±0.00), the mass-to-nucleus ratio of the synthesized peptide KP was 811.98 (±0.00), and the mass-to-nucleus ratio of the synthesized peptide PP was 904.90 (±0.00), all basically consistent with the theoretical molecular weight. The identification results indicate that the synthesized peptides were identified as RM, KP, and PP as required for the experiment.

[0092] Table 5. HPLC purity analysis of solid-phase synthesized nereisin RM, KP, and PP.

[0093] 3.2.4 Polysaccharidus bioactive peptides prolong the lifespan of wild-type and gene-deficient N2 nematodes. Following PA14 infection, the average lifespan of wild-type N2 nematodes was 9.51 days, a 33.41% reduction compared to the average lifespan of normal nematodes, with the longest lifespan decreasing by 6 days. The average lifespan of pmk-1 (km25) gene-deficient nematodes was 8.88 days, a 17.40% reduction compared to the CT group, with the longest lifespan decreasing by 3 days. The average lifespan of skn-1 (zu135) gene-deficient nematodes was 8.74 days, a 35.83% reduction compared to the average lifespan, with the longest lifespan decreasing by 3 days. When all three nematode species were immunosuppressed, their lifespan curves showed a significant leftward shift compared to the blank control group. Figure 12 (a) The lifespans of the two gene-deficient nematodes (km25 and zu135) were shortened by 6.62% and 8.10% respectively compared with the N2 wild-type nematode, and their maximum lifespans were shortened by 4 days and 3 days respectively. This indicates that the defects of these two genes lead to the shortening of the nematode lifespan, and thus it is inferred that the regulation of these two genes plays a very important role in prolonging the lifespan of nematodes and repairing immune damage.

[0094] Treatment with different concentrations of the nematode bioactive peptide RM prolonged the lifespan of wild-type N2 nematodes to varying degrees. The RM intervention at 10 μg / mL showed the best effect in extending both the average and maximum lifespan of the nematodes, with an average lifespan increase of 15.54% compared to the PA14-infected MD group. This indicates that the nematode bioactive peptide RM can effectively alleviate the immune damage caused by PA14 infection in wild-type N2 nematodes and reduce the toxic effects of PA14 on the nematodes.

[0095] Treatment of km25 and zu135 gene-deficient nematodes with RM significantly extended the lifespan of nematodes at all concentrations, with the 10 μg / mL concentration showing the best effect on both the average and maximum lifespan, resulting in a marked rightward shift in the lifespan curve compared to the MD group. Figure 12 In (b, c) the longest lifespan was extended, indicating that the active peptides KP and RM of Nematodea can enhance the resistance of pmk-1 (km25) and skn-1 (zu135) mutants to PA14 infection, improve the immune damage of nematodes, and regulate immune function through the transcription factors PMK-1 and SKN-1.

[0096] 3.2.5 Nematode bioactive peptides reduce lipofuscin accumulation in wild-type N2 nematodes. Further research was conducted on the improvement of immune-related health signs in nematodes by nervone active peptides. The accumulation of lipofuscin can reflect the immune status of nematodes to some extent. Lipofuscin is composed of lysosomal digestion products and fatty residues, and it can fluoresce at specific wavelengths. As the lifespan of nematodes shortens, lipofuscin gradually accumulates, and the fluorescence deepens. After N2 wild-type nematodes were infected with PA14, treatment with nervone active peptides KP, RM, and PP showed a concentration-dependent effect on lipofuscin accumulation between 1-10 μg / mL. As the concentration increased, lipofuscin accumulation decreased, with the greatest reduction observed at a concentration of 10 μg / mL. Compared to the MD group, the three nervone active peptides significantly reduced lipofuscin accumulation in nematodes. P<0.00 1)( Figure 13 In the image representing lipofuscin (b), its fluorescence intensity gradually decreases. Figure 13 (a) However, after treatment with 20 μg / mL, lipofuscin accumulation in nematodes increased, and their lifespan was shortened. This may be due to factors such as the toxic effects induced by high doses of bioactive peptides and increased oxidative stress, leading to increased lipofuscin accumulation and shortened lifespan. The results indicate that the bioactive peptides of *Nematodes nereis* enhance the immunity of nematodes, reduce the damage caused by infection, thereby improving the health status of the organism, prolonging the lifespan of nematodes, and ultimately reducing the accumulation of lipofuscin.

[0097] 3.2.6 Nematode bioactive peptides reduce lipofuscin accumulation in gene-deficient nematodes The above studies have shown that transcription factors PMK-1 and SKN-1 are involved in the regulation of nematode lifespan. To further investigate whether transcription factors PMK-1 and SKN-1 are involved in the immunomodulatory effects of PA14 infection on immunosuppressive nematodes, this invention examined the effect of the active peptide RM of *Nereidum nervosa* on lipofuscin accumulation in nematodes with deficient pmk-1 (km25) and skn-1 (zu135) genes. The results are as follows: Figure 14 and 15 As shown.

[0098] At a low concentration of 1 μg / mL, RM had no significant effect on pmk-1 (km25) gene-deficient nematodes. At concentrations of 5-10 μg / mL, compared to the model group, the fluorescence intensity in nematodes gradually decreased with increasing concentration, and the accumulation of lipofuscin gradually decreased. The effect of RM on reducing lipofuscin in nematodes was best at 10 μg / mL. Figure 14 (b)). For example Figure 14 As shown in (a), lipofuscin exhibits the weakest fluorescence intensity at a concentration of 10 μg / mL.

[0099] Treatment of skn-1(zu135) gene-deficient nematodes with RM active peptides at concentrations of 1–10 μg / mL significantly reduced lipofuscin accumulation compared to the MD group, with a marked decrease at 10 μg / mL. Figure 15 (b)). For example Figure 15 As shown in (a), the representative lipofuscin fluorescence image of skn-1 (zu135) gene-deficient nematodes also illustrates the above situation.

[0100] Studies have found that under PA14 infection conditions, the transcription factor PMK-1 promotes the nuclear translocation of SKN-1 by phosphorylating it, thereby enhancing the nematode's resistance to PA14, reducing oxidative stress-induced cell damage, effectively scavenging free radicals, and reducing lipofuscin accumulation. Conversely, silencing the PMK-1 and SKN-1 genes prevents the activation of downstream immune genes, leading to decreased expression levels of immune and antioxidant genes. This weakens the nematode's immune defense capabilities, exacerbates oxidative damage, and promotes lipofuscin accumulation.

[0101] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An immunologically active peptide derived from *Pterygodium bispinosa*, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The use of the immunomodulatory peptide as described in claim 1 in the preparation of products that enhance immunity.

3. A product for enhancing immunity, characterized in that, Includes the immunoactive peptide of claim 1.

4. The use of the immunomodulatory peptide as described in claim 1 in the preparation of products that extend lifespan.

5. A product with extended lifespan, characterized in that, Includes the immunoactive peptide of claim 1.

6. The use of the immunomodulatory peptide as described in claim 1 in the preparation of products that enhance reproductive capacity.

7. A product for improving reproductive capacity, characterized in that, Includes the immunoactive peptide of claim 1.

8. The use of the immunomodulatory peptide as described in claim 1 in the preparation of a product that inhibits the accumulation of lipofuscin.

9. A product for inhibiting the accumulation of lipofuscin, characterized in that, Includes the immunoactive peptide of claim 1.

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