Fish-derived antibacterial peptide as well as preparation method and application thereof

Abstract

The invention discloses a fish-derived antibacterial peptide as well as a preparation method and application thereof, the antibacterial peptide is derived from fish-derived Piscid-1 and Hepcidin, and the antibacterial peptide sequentially comprises a signal peptide, an antibacterial peptide, tag protein and other functional elements from an N end to a C end. According to the invention, by optimizing the connection method and sequence of fish-derived Piscid-1, Hepcidin, signal peptide, tag protein and other functional elements in the fish-derived antibacterial peptide, a plurality of engineering bacteria for expressing monomer fish-derived antibacterial peptide and tandem fish-derived antibacterial peptide are obtained, and optimal biological activity and function synergy are realized. The fish-derived antibacterial peptide and the engineering bacteria thereof can be used as feed additives to be applied to fish culture, and can play roles in promoting growth, enhancing immune performance, improving intestinal flora and the like. The method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology technology, and relates to a fish-derived antimicrobial peptide, its preparation method, and its application. Background Technology

[0002] Antimicrobial peptides (AMPs) are a class of small molecule polypeptides with broad-spectrum antimicrobial activity. They are considered ideal alternatives to traditional antibiotics because they are less likely to induce bacterial resistance. To date, more than 3,000 antimicrobial peptides have been isolated and identified in domestic and international research. These antimicrobial peptides come from a wide range of sources, including plants, insects, mammals, amphibians, aquatic animals, birds, bacteria, viruses, microbial engineering, and artificial synthesis.

[0003] Fish antimicrobial peptides are important non-specific immune factors that rapidly defend against and kill invading substances when attacked by pathogenic microorganisms. They are an important part of the fish's innate immune system. Based on their amino acid composition and structure, marine fish antimicrobial peptides can be classified into Piscidin, Defensin, Hepcidin, Cathelicidin, and Pardaxin. Among them, Piscidin is rich in histidine and phenylalanine, has an α-helical structure, and has been shown to have antibacterial, antifungal, antiparasitic, and antiviral activities [1]. Hepcidin has about 26 to 85 amino acids and has antibacterial activity against Gram-positive and Gram-negative bacteria [2]. Both types of fish-derived antimicrobial peptides have shown excellent antibacterial effects and participate in the body's immune response [3, 4]. They have significant scientific value, market demand, and development prospects, but there is still very little research and patents on them.

[0004] At present, the main ways to obtain antimicrobial peptides include natural extraction, chemical synthesis and gene engineering expression. Among them, recombinant expression using engineered bacteria has become a research hotspot due to its controllable cost and ease of large-scale production. However, the current mainstream expression systems have technical obstacles such as insufficient yield (Chinese patent document CN115505616A), loss of activity[5], host toxicity (Chinese patent document CN110093393A), easy degradation, and insufficient maturity of emerging systems (lactic acid bacteria / Bacillus subtilis), which leads to limited common use of antimicrobial peptides. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a fish-derived antimicrobial peptide, its preparation method, and its application, specifically relating to the heterologous expression of fish-derived antimicrobial peptides by probiotics and its in vivo induction expression method.

[0006] In one specific embodiment of this invention, the objective is to utilize Bacillus subtilis to secrete and express fish-derived antimicrobial peptides, and to achieve induced expression in the fish gut. This includes the construction of the inducible expression vector, the preparation method of the expression product, and the in vivo induction method of the engineered bacteria.

[0007] The innovation of this invention lies in the fact that while Piscidin-1 and Hepcidin from tilapia have been studied for their excellent antibacterial activity, there is little research or invention on their expression and delivery using probiotics as a substrate. This invention optimizes the connection methods and sequences of various signal peptides, Piscidin-1, Hepcidin, tag proteins, and other functional elements to obtain various monomeric and tandem fish-derived antimicrobial peptide proteins. Furthermore, the tandem fish-derived antimicrobial peptide fusion protein achieves optimal bioactivity and functional synergy. Simultaneously, by utilizing probiotics as a substrate, the above-mentioned fish-derived antimicrobial peptides are heterologously expressed, achieving a synergistic effect of probiotic and antimicrobial activity.

[0008] In this invention, the gene fragment "ph-his" refers to the linking of two antimicrobial peptide gene sequences (pisidin-1 gene sequence and hepcidin gene sequence) using the nucleotide sequence of the enterokinase site (enterokinase site sequence), with a 6×his tag added to the 3' end. Codon optimization was performed based on the codon preference of Bacillus subtilis (website: JCat) to obtain the gene fragment, named ph. The tandem protein "PH-his" refers to the linking of two antimicrobial peptide amino acid sequences (Piscidin-1 amino acid sequence and Hepcidin amino acid sequence) using the enterokinase site, with a 6×His tag protein added to the C-terminus.

[0009] This invention provides a fish-derived antimicrobial peptide, which is derived from fish-derived Piscidin-1, Hepcidin, etc., and includes a signal peptide, an antimicrobial peptide, a tag protein, and other functional elements from the N-terminus to the C-terminus.

[0010] Furthermore, the fish-derived antimicrobial peptides include recombinant antimicrobial peptides, etc.

[0011] Furthermore, the fish-derived antimicrobial peptides include one or more of monomeric fish-derived antimicrobial peptides and tandem fish-derived antimicrobial peptides.

[0012] Furthermore, the signal peptide and the antimicrobial peptide also include a linker, wherein the linker includes G4S, etc.

[0013] Furthermore, the signal peptide includes one or more of the following: Bacillus subtilis signal peptides AprE, AmyQ, SacB, and unconventional signal peptides sfGFP.

[0014] Furthermore, the antimicrobial peptide includes one or more of fish-derived Piscidin-1, Hepcidin, etc.

[0015] Furthermore, the tag protein includes one or more of 6×His, etc.

[0016] Furthermore, when the fish-derived antimicrobial peptides are formed by fish-derived Piscidin-1 and / or Hepcidin in tandem, the individual antimicrobial peptides also include enterokinase site sequences, etc.

[0017] Further, the fish-derived antimicrobial peptide includes one or more of the following proteins: AprE-Pisidin-1, AmyQ-Pisidin-1, SacB-Pisidin-1, sfGFP-Pisidin-1, AprE-Hepcidin, AmyQ-Hepcidin, SacB-Hepcidin, sfGFP-Hepcidin, and sfGFP-PH-his. Preferably, the fish-derived antimicrobial peptide includes sfGFP-PH-his, sfGFP-Piscidin-1, and sfGFP-Hepcidin.

[0018] The sfGFP-PH-his includes, from N-terminus to C-terminus, sfGFP, G4S, Piscidin-1, enterokinase site, Hepcidin, 6×His, etc.

[0019] The present invention also provides a polynucleotide, including polynucleotides encoding fish-derived antimicrobial peptides as described above.

[0020] Furthermore, the polynucleotides include aprE-pisidin-1, amyQ-pisidin-1, sacB-pisidin-1, sfgfp-pisidin-1, etc.

[0021] Furthermore, the polynucleotides include aprE-hepcidin, amyQ-hepcidin, sacB-hepcidin, sfgfp-hepcidin, etc.

[0022] Furthermore, the polynucleotides include sfgfp-ph-his, etc.

[0023] The sfgfp-ph-his sequence includes sfgfp, linker g4s, piscidin-1, enterokinase site sequence, hepcidin, and 6×his from the 5' end to the 3' end.

[0024] The present invention also provides a recombinant vector, which includes polynucleotides as described above.

[0025] Furthermore, the carrier includes non-viral carriers, etc.

[0026] Non-viral vectors include plasmid vectors, among others. These plasmid vectors include cloning vectors and expression vectors.

[0027] Furthermore, the recombinant vector includes an inducible promoter PxylA, structural genes (with signal peptides linked sequentially from 5' to 3', antimicrobial peptide genes (one or more of piscidin-1, hepcidin, etc.), tag proteins, etc.), and other basic plasmid elements.

[0028] In one specific embodiment, the recombinant vector includes an inducible promoter PxylA, structural genes (5' to 3' sequentially linked to sfgfp, linker g4s, antimicrobial peptide gene piscidin-1, enterokinase site sequence, antimicrobial peptide gene hepcidin, tag protein, etc.), and other basic plasmid elements.

[0029] In one specific embodiment, the inducible promoter PxylA is derived from the xyloperon of Bacillus megaterium and includes the core promoter region and upstream regulatory sequences. In the absence of xylose, the XylR protein binds to the PxylA operon, blocking RNA polymerase binding, resulting in extremely low background expression (high rigor). In the presence of xylose, xylose binds to the XylR protein, causing a conformational change that dissociates it from the operon, thus releasing promoter repression and significantly improving transcription efficiency (induction ratio can reach >100-fold). Furthermore, xylose is safe and non-toxic (GRAS certified), and its cost is lower than IPTG, making it suitable for food-grade applications.

[0030] Furthermore, the recombinant vector includes one or more of pSTOP1622-aprE-pisidin-1, pSTOP1622-amyQ-pisidin-1, pSTOP1622-sacB-pisidin-1, and pSTOP1622-sfgfp-pisidin-1.

[0031] Furthermore, the recombinant vector includes one or more of pSTOP1622-aprE-hepcidin, pSTOP1622-amyQ-hepcidin, pSTOP1622-sacB-hepcidin, and pSTOP1622-sfgfp-hepcidin.

[0032] Furthermore, the recombinant vector includes pSTOP1622-sfgfp-ph-his, etc.

[0033] The present invention also provides a transformant, which includes one or more of the polynucleotides described above or the recombinant vectors described above.

[0034] Furthermore, the transformant includes Bacillus subtilis, etc. Furthermore, the transformant includes Bacillus subtilis BS168, etc.

[0035] In one specific embodiment, the present invention uses Bacillus subtilis BS168 as the chassis cell to provide multiple engineered bacteria expressing fish-derived antimicrobial peptides, including BS168-AprE-Pisidin-1, BS168-AmyQ-Pisidin-1, BS168-SacB-Pisidin-1, BS168-sfGFP-Pisidin-1, BS168-AprE-Hepcidin, BS168-AmyQ-Hepcidin, BS168-SacB-Hepcidin, BS168-sfGFP-Hepcidin, and BS168-sfGFP-PH-his. Bacillus subtilis is certified as a "Generally Recognized as Safe" (GRAS) microorganism by the U.S. Food and Drug Administration (FDA), meaning it is non-pathogenic, does not produce endotoxins, and meets the stringent regulatory requirements for pharmaceutical and food-grade production. Furthermore, Bacillus subtilis is characterized by high secretion efficiency, low fermentation cost, and mature genetic manipulation tools. Importantly, the "integrated microbial-agent" model of Bacillus subtilis can be directly applied to feed and agriculture. Its live bacteria can be directly used as a feed additive to improve intestinal flora and immune response; simultaneously, engineered bacteria using Bacillus subtilis as a substrate can secrete active substances such as chitinase, providing disease control and other functions, achieving "multiple uses from one strain." The whole genome of strain BS168 was sequenced in 1997, and its clear genetic background provided a foundation for gene editing. The fermentation residue of BS168 contains live bacteria and antimicrobial peptides, which can be directly used in livestock and poultry feed, such as inhibiting Streptococcus suis.

[0036] In one specific embodiment of this invention, the engineered bacteria expressing Piscidin-1 monomers all exhibited significant inhibitory effects against Aeromonas hydrophila in vitro. Among them, the engineered bacteria BS168-sfGFP-Pisidin-1, using the unconventional signal peptide sfGFP, showed the best inhibitory effect.

[0037] In one specific embodiment of this invention, the engineered bacteria BS168-AprE-Hepcidin and BS168-sfGFP-Hepcidin exhibit significant inhibitory effects against Aeromonas hydrophila in vitro. Among them, the engineered bacteria BS168-sfGFP-Hepcidin, which uses the unconventional signal peptide sfGFP, shows the best inhibitory effect.

[0038] In one specific embodiment of the present invention, the BS168-sfGFP-PH-his is directly delivered to the zebrafish intestine, which can efficiently secrete recombinant antimicrobial peptides and exert its probiotic effect while ensuring safety.

[0039] This invention is the first to express fish-derived Piscidin-1 and Hepcidin in tandem, and precisely control the expression sequence using a xylose induction system.

[0040] In one specific embodiment, the present invention provides a method for secreting and expressing various fish-derived antimicrobial peptide monomers, Piscidin-1, which includes adding different signal peptides to the N-terminus of the antimicrobial peptide Piscidin-1 protein to guide the secretory expression of the monomeric antimicrobial peptide protein.

[0041] In one specific embodiment, the present invention provides a method for secreting and expressing various fish-derived antimicrobial peptide monomers, namely Hepcidin, which includes adding different signal peptides to the N-terminus of the antimicrobial peptide Hepcidin protein to guide the secretory expression of the monomeric antimicrobial peptide protein.

[0042] The present invention also provides a method for preparing the fish-derived antimicrobial peptide as described above, the method comprising the following steps:

[0043] The first step is to construct the recombinant vector as described above;

[0044] The second step is to transform the recombinant vector constructed in the first step into the host to obtain the transformed organism (the transformed host);

[0045] The third step is to express the transformant obtained in the second step to prepare the expression product, namely the fish-derived antimicrobial peptide as described above.

[0046] Furthermore, the preparation method also includes verification of the fish-derived antimicrobial peptide obtained in the third step.

[0047] Furthermore, the preparation method also includes purifying the fish-derived antimicrobial peptide obtained in the third step.

[0048] In one specific embodiment, the recombinant vector includes pSTOP1622-aprE-pisidin-1, pSTOP1622-amyQ-pisidin-1, pSTOP1622-sacB-pisidin-1, pSTOP1622-sfgfp-pisidin-1, pSTOP1622-aprE-hepcidin, pSTOP1622-amyQ-hepcidin, pSTOP1622-sacB-hepcidin, pSTOP1622-sfgfp-hepcidin, and pSTOP16 The transformant comprises one or more of BS168-AprE-Pisidin-1, BS168-AmyQ-Pisidin-1, BS168-SacB-Pisidin-1, BS168-sfGFP-Pisidin-1, BS168-AprE-Hepcidin, BS168-AmyQ-Hepcidin, BS168-SacB-Hepcidin, BS168-sfGFP-Hepcidin, and BS168-sfGFP-PH-his. The fish-derived antimicrobial peptides include one or more of the following: AprE-Pisidin-1, AmyQ-Pisidin-1, SacB-Pisidin-1, sfGFP-Pisidin-1, AprE-Hepcidin, AmyQ-Hepcidin, SacB-Hepcidin, sfGFP-Hepcidin, and sfGFP-PH-his protein.

[0049] In a preferred embodiment, the recombinant vector includes pSTOP1622-sfgfp-ph-his, pSTOP1622-sfgfp-piscidin-1, pSTOP1622-sfgfp-hepcidin, etc., and the transformants include BS168-sfGFP-PH-his, BS168-sfGFP-Piscidin-1, BS168-sfGFP-Hepcidin, etc. The fish-derived antimicrobial peptides include sfGFP-PH-his, sfGFP-Piscidin-1, sfGFP-Hepcidin proteins, etc. Wherein, sfGFP is an unconventional signal peptide.

[0050] The present invention also provides an in vivo induction method for the transformant as described above, the induction method comprising: adding the transformant as described above to animal feed, and simultaneously adding an inducer for in vivo induction.

[0051] Further, the animals include farmed animals, etc. Further, the animals include aquaculture animals, etc. Further, the animals include aquaculture fish, etc. Further, the animals include zebrafish, tilapia, etc. Preferably, the animals include zebrafish, etc.

[0052] Furthermore, the transformant includes one or more of the engineered bacteria BS168-AprE-Pisidin-1, BS168-AmyQ-Pisidin-1, BS168-SacB-Pisidin-1, BS168-sfGFP-Pisidin-1, BS168-AprE-Hepcidin, BS168-AmyQ-Hepcidin, BS168-SacB-Hepcidin, BS168-sfGFP-Hepcidin, and BS168-sfGFP-PH-his.

[0053] Preferably, the transformant includes one or more of the engineered bacteria BS168-sfGFP-Piscidin-1, BS168-sfGFP-Hepcidin, BS168-sfGFP-PH-his, etc.

[0054] Furthermore, the recombinant vector contained in the transformant includes recombinant vectors that do not contain resistance gene tags, etc.

[0055] Furthermore, the concentration of the transformant is 10. 5 -10 10 CFU / g; preferably, 10. 8 CFU / g. Further, the inducing agent includes xylose, etc.

[0056] The concentration of the inducer is 0.5%-5%; preferably, it is 1.5%.

[0057] Furthermore, the induction method also includes setting up animals fed with the in vitro induced transformant and animals without the induced transformant as controls.

[0058] In one specific embodiment, the induction method is as follows: adding uninduced engineered bacteria BS168-sfGFP-PH-his to zebrafish feed (10 8 The strain was formulated with CFU / g and 1.5% xylose for in vivo induction. Zebrafish fed with the engineered bacteria induced in vitro for 12 hours and those without the engineered bacteria were used as controls. Six hours after feeding, the zebrafish intestines were collected and observed under a fluorescence microscope. Fluorescence microscopy revealed obvious green fluorescence in the intestines of the in vivo-induced zebrafish, demonstrating that the engineered bacteria BS168-sfGFP-PH-his can achieve in vivo induction.

[0059] This invention also proposes the application of the in vivo induction method described above in fish farming and other fields.

[0060] Specifically, the in vivo induction method described above allows for precise control of the timing of recombinant antimicrobial peptide expression and secretion during fish farming. When farmed fish are infected by pathogens or experience other environmental stress during their growth, adding xylose as an inducer to the feed can induce a rapid response in the engineered bacteria in the fish's gut, causing them to synthesize and secrete recombinant antimicrobial peptides, thereby killing pathogens and improving immunity. Furthermore, for smaller fish or early-stage juveniles, the success of in vivo induction can be determined non-invasively by observing the fluorescence of sfGFP, thus saving farming costs.

[0061] In one specific embodiment of this invention, zebrafish were used as a model. Zebrafish were infected with *Aeromonas hydrophila*, and xylose (1.5%) was added to their feed as an inducer. This induced the engineered bacteria BS168-sfGFP-PH-his colonizing the intestines to synthesize and secrete the recombinant antimicrobial peptide sfGFP-PH-his. This in vivo induction method effectively salvaged the survival rate of zebrafish challenged with *Aeromonas hydrophila*, reduced the level of *Aeromonas hydrophila* in the intestines, and improved the inflammatory environment of the zebrafish intestines.

[0062] The present invention provides a novel drug / drug composition comprising one or more of the polynucleotides, recombinant vectors, or transformants as described above; preferably, the drug / drug composition comprises transformants as described above.

[0063] In one specific embodiment, the drug includes engineered bacteria such as BS168-sfGFP-PH-his, which can deliver antimicrobial peptides to the animal's intestines. Bacillus subtilis is certified as a "Generally Recognized as Safe" (GRAS) microorganism by the U.S. Food and Drug Administration (FDA), meeting the stringent regulatory requirements for pharmaceutical and food-grade production. The engineered bacteria BS168-sfGFP-PH-his, without pathogenicity, kills pathogens, improves the inflammatory environment, and stimulates the immune response, thereby improving the pathological state by secreting recombinant antimicrobial peptides.

[0064] In one specific embodiment of the present invention, the engineered bacterium BS168-sfGFP-PH-his effectively improved the pathological state caused by Aeromonas hydrophila infection, such as elevated pro-inflammatory factors and damaged intestinal structure, thus saving the survival rate of zebrafish.

[0065] This invention provides a novel feed additive, comprising one or more of the following: polynucleotides as described above, recombinant vectors as described above, or transformants as described above. Furthermore, the feed additive also contains inducers, etc.

[0066] In one specific embodiment, the feed additive includes the engineered bacteria BS168-sfGFP-PH-his or the engineered bacteria BS168-sfGFP-PH-his / inducer xylose combination as described above.

[0067] In one specific embodiment, the feed additive is suitable for farmed fish, including but not limited to zebrafish, tilapia, and other aquaculture fish. Preferably, the target is zebrafish. Specifically, the engineered bacteria BS168-sfGFP-PH-his in the feed additive can promote growth, enhance immune performance, and improve intestinal flora. After the addition is stopped, the engineered bacteria can be completely metabolized and excreted within 10-14 days, avoiding any lasting impact on the fish's intestines. Using the combination of the engineered bacteria BS168-sfGFP-PH-his in the feed additive and the xylose inducer, the engineered bacteria can continuously secrete recombinant antimicrobial peptides in the fish's intestines to kill pathogens, improve intestinal immunity, and protect intestinal structure.

[0068] The present invention also proposes the application of the feed additives described above in improving environmental pollution and other aspects.

[0069] Specifically, the feed additives described above have a simple production process, low energy consumption, and relatively low greenhouse gas emissions. Furthermore, all feed additives described above are environmentally friendly materials that can be naturally degraded. This can reduce / replace the use of high-carbon-footprint drugs and mitigate chemical pollution (active drug residues), biological pollution (ARGs / pathogens), ecological imbalance (critical species extinction), and health threats (drug-resistant infections) caused by the multi-media migration of traditional chemical drugs.

[0070] In one specific embodiment, the recombinant antimicrobial peptides secreted by the feed additive BS168-sfGFP-PH-his / inducer xylose combination can be degraded by proteases in the zebrafish intestine without leaving any tissue residue; in addition to being utilized by engineered bacteria as an inducer, xylose can be directly metabolized by the fish and can also indirectly regulate intestinal homeostasis as a prebiotic; the engineered bacteria BS168-sfGFP-PH-his has strong intestinal safety and can be completely metabolized and excreted within 10-14 days after feeding is stopped.

[0071] This invention provides a novel probiotic preparation, which includes the transformants as described above.

[0072] Specifically, this probiotic preparation can be directly applied to inhibit pathogens in vivo and in vitro, reducing the process of extracting and purifying recombinant antimicrobial peptides and lowering production costs.

[0073] In one specific embodiment, the probiotic preparation includes BS168-sfGFP-PH-his, etc., which, when applied to zebrafish infected with Aeromonas hydrophila, effectively reduced the Aeromonas hydrophila load in the zebrafish intestine and saved their survival rate.

[0074] The present invention also provides a fermentation and concentration method for the probiotic preparation as described above, the fermentation and concentration method comprising: preparing seeds, expanding culture, fermentation culture, washing and concentration, etc.

[0075] Specifically, the fermentation and concentration method includes the following steps:

[0076] The first step is seed preparation: this involves inoculating a single colony of the transformant as described above into fresh liquid culture medium and culturing at 37°C and 200 rpm for 8-12 hours to obtain seeds. The second step is scale-up culture: this involves inoculating the seeds obtained in the first step into fresh liquid culture medium at a concentration of 1.5%-5% and culturing at 37°C and 200 rpm for 8-12 hours to obtain scale-up culture products. The third step is fermentation culture: this involves adding 0.5%-5% xylose, an inducer, to the scale-up culture products obtained in the second step and culturing for 12-36 hours to obtain fermentation culture products. The fourth step is washing and concentration: this involves collecting the bacterial cells and fermentation supernatant from the fermentation culture products obtained in the third step by refrigerated centrifugation (4°C, 6000×g, 20 min), and then resuspending the bacterial cells in PBS (phosphate buffered saline) to obtain the probiotic preparation as described above.

[0077] In the fermentation and concentration method, the liquid culture medium includes LB (1% tryptone, 0.5% yeast extract, 1% NaCl, ddH2O, etc.) liquid culture medium, preferably LB liquid culture medium.

[0078] In one specific embodiment, the probiotic preparation includes BS168-sfGFP-Pisidin-1, BS168-sfGFP-Hepcidin, BS168-sfGFP-PH-his, etc. A concentration of 10 was prepared and obtained through the fermentation and concentration methods described above. 8 Three probiotic formulations at CFU / mL were applied to zebrafish infected with Aeromonas hydrophila. The formulation BS168-sfGFP-PH-his effectively improved the intestinal immune function of the zebrafish, thus improving their survival rate.

[0079] This invention provides a novel metabiotic, which includes, as described above, fish-derived antimicrobial peptides, etc.

[0080] Specifically, the metabiotic includes one or more of the following: monomeric fish-derived antimicrobial peptides secreted by engineered bacteria: AprE-Pisidin-1, AmyQ-Pisidin-1, SacB-Pisidin-1, sfGFP-Pisidin-1, AprE-Hepcidin, AmyQ-Hepcidin, SacB-Hepcidin, sfGFP-Hepcidin, and tandem fish-derived antimicrobial peptide sfGFP-PH-his protein.

[0081] Preferably, the metabiotic includes one or more of the following: monomeric fish-derived antimicrobial peptides secreted by engineered bacteria, such as sfGFP-Piscidin-1 and sfGFP-Hepcidin, and tandem fish-derived antimicrobial peptides such as sfGFP-PH-his.

[0082] In this invention, metabiotics refer to preparations of non-living microorganisms and / or their components that are beneficial to the health of the host, including metabolites (such as short-chain fatty acids, enzymes, and peptides) and cellular components (such as cell wall fragments). Bacillus subtilis is widely used as a probiotic in aquaculture. This invention utilizes synthetic biology methods to induce Bacillus subtilis BS168 to secrete recombinant fish-derived antimicrobial peptide monomers sfGFP-Piscidin-1, sfGFP-Hepcidin, and / or the tandem antimicrobial peptide sfGFP-PH-his. In the zebrafish gut, these metabiotics can exert beneficial effects such as regulating intestinal flora, antibacterial activity, anti-inflammation, and antioxidant activity.

[0083] The present invention also proposes the application of the proteins described above in antibiotic replacement (antibiotic alternatives), etc.

[0084] Specifically, recombinant antimicrobial peptides sfGFP-Piscidin-1 and sfGFP-Hepcidin have the potential to serve as antibiotic alternatives. The bactericidal mechanism of antimicrobial peptides differs from that of traditional antibiotics, making them less prone to inducing resistance. Furthermore, in the animal gut, antimicrobial peptides can be degraded by proteases, leaving no residue risk, making them suitable for replacing subtherapeutic dose antibiotics in livestock farming.

[0085] In one specific embodiment, the recombinant antimicrobial peptides sfGFP-Piscidin-1 and sfGFP-Hepcidin both showed significant inhibitory effects on Aeromonas hydrophila in vitro, and had similar antibacterial effects to the positive control ampicillin.

[0086] The present invention also proposes a method for preventing / inhibiting / alleviating / reducing / treating diseases, the method comprising administering to a subject in need an effective amount of one or more of the following: fish-derived antimicrobial peptides as described above, or polynucleotides as described above, or recombinant vectors as described above, or transformants as described above, or pharmaceuticals / pharmaceutical compositions as described above, or feed additives as described above, or probiotic preparations as described above, or postbiotics as described above.

[0087] In one specific embodiment, the target or individual for prevention / inhibition / alleviation / reduction / treatment is preferably fish, including but not limited to zebrafish, tilapia, and other aquaculture fish. More preferably, the target or individual is zebrafish. Specifically, the target can be zebrafish infected with Aeromonas hydrophila or zebrafish at risk of infection. One or more of the following can be administered to the target before, during, or after Aeromonas hydrophila infection: fish-derived antimicrobial peptides, polynucleotides, recombinant vectors, transformants, drugs / drug compositions, feed additives, probiotic preparations, or metabiotics. Specifically, before Aeromonas hydrophila infection, because Bacillus subtilis colonizes the fish gut for a short time, to maintain the level of engineered bacteria in the gut, engineered bacteria BS168-sfGFP-PH-his(10) (with resistance tag removed) can be added to the zebrafish basal feed for one week. 8 (CFU / g). In cases of Aeromonas hydrophila infection, xylose (1.5%) was added as an inducer while feeding engineered bacteria. This induced the engineered bacteria colonizing the intestine to synthesize and secrete the recombinant antimicrobial peptide sfGFP-PH-his, thereby reducing the level of Aeromonas hydrophila in the intestine and achieving a therapeutic effect. After Aeromonas hydrophila infection, the engineered bacteria can be fed again to inhibit pathogens, promote probiotics, and restore intestinal flora homeostasis.

[0088] In one specific embodiment of the present invention, compared with engineered bacteria BS168-sfGFP-Piscidin-1 and BS168-sfGFP-Hepcidin, engineered bacteria BS168-sfGFP-PH-his, which tandemly expresses Pisicidin-1 and Hepcidin, can effectively improve the survival rate of zebrafish challenged with Aeromonas hydrophila.

[0089] The present invention also provides a primer, which includes one or more of the following: sfGFP-PF, sfGFP-PR, PF, PR, 1622-F, 1622-R, HF, HR, PH-F, PH-R, SPST-F, SPST-R, AHaerA-F, AHaerA-R, Tnf-aF, Tnf-aR, il-1b-F, il-1b-R, Cxcl8-F, Cxcl8-R, il-6-F, il-6-R, il-10-F, il-10-R, il-22-F, il-22-R, Tgfb-F, and Tgfb-R.

[0090] The present invention also provides a primer pair, which includes one or more of the following: sfGFP-PF / sfGFP-PR, PF / PR, 1622-F / 1622-R, HF / HR, PH-F / PH-R, SPST-F / SPST-R, AHaerA-F / AHaerA-R, Tnf-aF / Tnf-aR, il-1b-F / il-1b-R, Cxcl8-F / Cxcl8-R, il-6-F / il-6-R, il-10-F / il-10-R, il-22-F / il-22-R, and Tgfb-F / Tgfb-R.

[0091] The present invention also provides a nucleotide, said nucleotide including one or more of enterokinase site sequences, linker g4s, etc.

[0092] The present invention also provides a polypeptide, wherein the polypeptide includes one or more of enterokinase sites, G4S, etc. Enterokinase sites and G4S are both commonly used functional elements in heterologous expression.

[0093] The present invention also provides an application of the unconventional signal peptide sfGFP in the heterologous expression of fish-derived antimicrobial peptides, the application comprising using the fusion protein sfGFP as a signal peptide at the N-terminus of the antimicrobial peptide to guide the secretion of the recombinant antimicrobial peptide into the extracellular space.

[0094] Superfolded green fluorescent protein (sfGFP) is obtained by six rounds of mutations (S30R, Y39N, N105T, Y145F, I171V, A206V) on GFP. Its folding rate and stability are significantly improved, reaching 3.5 times that of GFP. It is non-cytotoxic, has good transmissibility, and is widely used as a reporter gene in gene expression rate analysis, protein localization, cell localization, molecular interactions, cell screening, and biosensors. Furthermore, it emits 507nm green fluorescence directly under 488nm blue light excitation without the need for exogenous substrates, facilitating detection. However, studies have shown that certain proteins fused with sfGFP can undergo sfGFP-mediated transmembrane transport. [6 ].

[0095] This invention is the first to apply sfGFP as a signal peptide to the heterologous expression of fish-derived antimicrobial peptides, achieving secretory expression of recombinant antimicrobial peptides and ensuring the detectability of the expression process.

[0096] In one specific embodiment, compared with engineered bacteria using Bacillus subtilis signal peptides AprE, AmyQ, and SacB, using sfGFP as the signal peptide effectively increased the secretion efficiency of recombinant antimicrobial peptides. Figure 18 ).

[0097] Due to the spread of antibiotic resistance, drug residues, and environmental pollution, many countries have banned the use of antibiotics in aquaculture. Therefore, feed additives, as mentioned above, can play a role as antibiotic alternatives in combating pathogen infection and improving the intestinal health of farmed fish due to their characteristics of no residue, low risk of drug resistance, and ease of production.

[0098] This invention also proposes applications of the fish-derived antimicrobial peptides, polynucleotides, recombinant vectors, transformants, methods, drugs / drug compositions, feed additives, probiotic preparations, metabiotics, primers, primer pairs, nucleotides, and polypeptides as described above in antibiotic substitution, gut health of farmed fish, fish farming, promoting fish growth, combating pathogen infection, mitigating the spread of antibiotic resistance, reducing drug residues, mitigating environmental pollution, reducing the risk of drug resistance, improving intestinal immunity, protecting intestinal structure, improving the intestinal inflammatory environment, restoring intestinal flora homeostasis, preparing drugs for the prevention / inhibition / alleviation / reduction / treatment / diagnosis of diseases, developing feed additives, developing antimicrobial peptides, heterologous expression of fish-derived antimicrobial peptides, and saving aquaculture costs.

[0099] In this invention, G4S is a commonly used flexible linker that can avoid steric hindrance and maintain the activity of each domain. Fish-derived Piscidin-1 and Hepcidin have small molecular weights and are easily degraded by host proteases; tandem expression increases protein steric hindrance and reduces protease recognition efficiency. Furthermore, the mRNA of small peptides is easily degraded rapidly by host RNases. Tandem genes form longer open reading frames (ORFs), enhancing mRNA stability. By linking the two antimicrobial peptide genes using enterokinase sites, the recombinant antimicrobial peptide is secreted into the intestine, where it can be cleaved by enterokinase, releasing two monomers to exert their respective biological effects. In one specific embodiment, G4S and its nucleic acid sequence (linker g4s) are applied to the polynucleotides, recombinant vectors, and transformants described above, including sfgfp-ph-his, pSTOP1622-sfgfp-ph-his, BS168-sfGFP-PH-his, etc.

[0100] In this invention, the 6×His tag, composed of six consecutive histidine residues, is a commonly used tag protein with a molecular weight of only 0.84 kDa. It can interact with transition metal ions (Ni) via the imidazole ring of histidine. 2 +、Co 2+ Cu 2+ (e.g., forming coordination bonds) to achieve immobilized metal ion affinity chromatography. Adding a 6×his tag to the C-terminus avoids interference with functional domains and provides a foundation for subsequent purification of recombinant proteins.

[0101] In this invention, AprE-Pisidin-1, AmyQ-Pisidin-1, SacB-Pisidin-1, sfGFP-Pisidin-1, AprE-Hepcidin, AmyQ-Hepcidin, SacB-Hepcidin, and sfGFP-Hepcidin are monomeric fish-derived antimicrobial peptides; and / or, sfGFP-PH-his is a tandem fish-derived antimicrobial peptide.

[0102] In this invention, the amino acid sequence of the fish-derived antimicrobial peptide includes one of the following sequences:

[0103] (a) an amino acid sequence as shown in SEQ ID NO:1-9; (b) an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1-9; (c) an amino acid sequence having the same function but with one or more amino acid residues substituted, deleted, or added to the amino acid sequence shown in SEQ ID NO:1-9; (d) an amino acid fragment expressing the same function after substitution, deletion, or addition of one or more nucleic acid bases based on the coding nucleotide sequence of the amino acid sequence shown in SEQ ID NO:1-9; (e) an amino acid sequence having the same function that can hybridize with the coding nucleotide sequence of the amino acid sequence shown in SEQ ID NO:1-9 under moderately stringent conditions.

[0104] In this invention, the polynucleotide sequence includes one of the following sequences:

[0105] (a) a nucleotide sequence as shown in SEQ ID NO:10-18; (b) a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence defined in (a); (c) a nucleic acid sequence having equivalent function formed by substitution, deletion, or addition of one or more nucleic acid bases in the nucleotide sequence defined in (a); (d) a nucleotide sequence that, under stringent conditions, hybridizes with the nucleotide sequence defined in (a), (b), or (c) or its full-length complement; or, (e) a nucleotide sequence that is distinguished from the nucleotide sequences defined in (a), (b), (c), or (d) due to the degeneracy of the genetic codon;

[0106] In this invention, the nucleotide sequences of sfGFP-PF, sfGFP-PR, PF, PR, 1622-F, 1622-R, HF, HR, PH-F, PH-R, SPST-F, SPST-R, Tnf-aF, Tnf-aR, il-1b-F, il-1b-R, Cxcl8-F, Cxcl8-R, il-6-F, il-6-R, il-10-F, il-10-R, il-22-F, il-22-R, Tgfb-F, Tgfb-R, AHaerA-F, and AHaerA-R each include one of the following sequences:

[0107] (a) nucleotide sequences as shown in SEQ ID NO: 30-35 and 37-58, respectively; (b) nucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequence defined in (a); (c) nucleic acid sequences having equivalent function formed by substitution, deletion, or addition of one or more nucleic acid bases in the nucleotide sequence defined in (a); (d) nucleotide sequences that, under stringent conditions, hybridize with the nucleotide sequence defined in (a), (b), or (c) or its full-length complement; or, (e) nucleotide sequences that differ from the nucleotide sequences defined in (a), (b), (c), and (d) due to the degeneracy of the genetic codon.

[0108] In this invention, the nucleotide sequences include one of the following sequences:

[0109] (a) a nucleotide sequence as shown in SEQ ID NO: 22, 24; (b) a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence defined in (a); (c) a nucleic acid sequence having equivalent function formed by substitution, deletion, or addition of one or more nucleic acid bases in the nucleotide sequence defined in (a); (d) a nucleotide sequence that, under stringent conditions, hybridizes with the nucleotide sequence defined in (a), (b), or (c) or its full-length complement; or, (e) a nucleotide sequence that is distinguished from the nucleotide sequences defined in (a), (b), (c), or (d) due to the degeneracy of the genetic codon;

[0110] In this invention, the amino acid sequence of the polypeptide includes one of the following sequences:

[0111] (a) The amino acid sequences shown in SEQ ID NO:21 and 23; (b) Amino acid sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in SEQ ID NO:21 and 23; (c) Amino acid sequences having the same function but with one or more amino acid residues substituted, deleted, or added to the amino acid sequences shown in SEQ ID NO:21 and 23; (d) Sequences of amino acid fragments expressing the same function after substitution, deletion, or addition of one or more nucleic acid bases based on the coding nucleotide sequence of the amino acid sequences shown in SEQ ID NO:21 and 23; (e) Amino acid sequences having the same function that can hybridize with the coding nucleotide sequence of the amino acid sequences shown in SEQ ID NO:21 and 23 under moderately stringent conditions.

[0112] In this invention, the nucleotide sequence of sfGFP is shown in SEQ ID NO: 29; the nucleotide sequence of pisidin-1 is shown in SEQ ID NO: 25; the nucleotide sequence of hepcidin is shown in SEQ ID NO: 36; the amino acid sequence of 6×His is shown in SEQ ID NO: 19; the nucleotide sequence of 6×His is shown in SEQ ID NO: 20; the nucleotide sequence of aprE is shown in SEQ ID NO: 26; the nucleotide sequence of amyQ is shown in SEQ ID NO: 27; the nucleotide sequence of sacB is shown in SEQ ID NO: 28; and sfGFP is an unconventional signal peptide.

[0113] In this invention, the diseases include pathogenic bacterial infection, intestinal inflammation, intestinal tissue damage, intestinal flora imbalance, environmental stress, etc.

[0114] In one specific embodiment of the present invention, the disease includes an increase in pathogen load, intestinal inflammation, intestinal tissue damage, or individual death caused by infection with Aeromonas hydrophila.

[0115] Compared with existing technologies, the beneficial effects of this invention are reflected in: live bacteria delivery, reducing the loss of recombinant antimicrobial peptide activity and achieving the superposition of probiotic and antimicrobial effects; inducible expression, reducing host toxicity and achieving precise regulation of recombinant antimicrobial peptide synthesis and secretion; no residue and low risk of drug resistance; no need for purification, reducing production costs; effectively killing pathogens in the zebrafish intestine, improving immune performance, protecting intestinal health, and improving the survival rate of zebrafish. Attached Figure Description

[0116] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0117] Figure 1 The image shows the expression vectors of the monomeric antimicrobial peptide Piscidin-1, from left to right: pSTOP1622-aprE-pisidin-1, pSTOP1622-amyQ-pisidin-1, pSTOP1622-sacB-pisidin-1, and pSTOP1622-sfgfp-pisidin-1.

[0118] Figure 2 The plate confrontation experiment between engineered bacteria BS168-AprE-Pisidin-1, BS168-AmyQ-Pisidin-1, BS168-SacB-Pisidin-1, and BS168-sfGFP-Pisidin-1 and Aeromonas hydrophila, and the results of their inhibition zone determination.

[0119] Figure 3 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the recombinant antimicrobial peptide protein sfGFP-Piscidin-1. Lanes 7-14 were for the target protein eluted with protein elution buffer.

[0120] Figure 4 To determine the antibacterial activity of the recombinant antimicrobial peptide protein sfGFP-Piscidin-1, ampicillin (AMP+) was used as a positive control and PBS as a negative control. TIME represents time (in hours).

[0121] Figure 5 The image shows the expression vectors of the monomeric antimicrobial peptide Hepcidin, from left to right: pSTOP1622-aprE-hepcidin, pSTOP1622-amyQ-hepcidin, pSTOP1622-sacB-hepcidin, and pSTOP1622-sfgfp-hepcidin.

[0122] Figure 6The plate confrontation experiment between engineered bacteria BS168-AprE-Hepcidin, BS168-AmyQ-Hepcidin, BS168-SacB-Hepcidin, and BS168-sfGFP-Hepcidin and Aeromonas hydrophila, and the results of their inhibition zone determination.

[0123] Figure 7 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the recombinant antimicrobial peptide protein sfGFP-Hepcidin. Lanes 7-10 were for the target protein eluted with protein elution buffer.

[0124] Figure 8 This study determined the antibacterial activity of the recombinant antimicrobial peptide sfGFP-Hepcidin. Ampicillin (AMP+) was used as a positive control, and PBS as a negative control. TIME represents time (in hours).

[0125] Figure 9 The image shows the recombinant plasmid pSTOP1622-sfgfp-ph-his.

[0126] Figure 10 Agarose gel electrophoresis was performed on the amplified plasmid pSTOP1622-sfGFP-PH-his fragment using primers SPST-F / SPST-R.

[0127] Figure 11 The colony morphology of the engineered bacterium BS168-sfGFP-PH-his under natural light and blue light.

[0128] Figure 12 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on the recombinant antimicrobial peptide protein sfGFP-PH-his. Lanes 3-5 and 12-15 were used for eluting the target protein with protein elution buffer.

[0129] Figure 13 This is a fluorescence image of engineered bacteria in the intestine of zebrafish. From top to bottom, the intestines are derived from zebrafish induced in vitro, zebrafish induced in vivo by xylose, and uninduced zebrafish.

[0130] Figure 14 This data represents the survival rate of zebrafish challenged with Aeromonas hydrophila. Wherein, Probability of Survival: survival rate; DAY: number of days.

[0131] Figure 15 This represents the absolute quantification of Aeromonas hydrophila in the intestinal contents of zebrafish. Where copies / mg: copies per milligram.

[0132] Figure 16Real-time quantitative PCR (qPCR) of pro-inflammatory factors in zebrafish intestinal tissue. From left to right: tnfa, il-1β, cxcl8, il-6. Relative mRNA expression levels are: tnfa, il-1β, cxcl8, and il-6.

[0133] Figure 17 Real-time quantitative PCR (qPCR) of anti-inflammatory factors in zebrafish intestinal tissue. From left to right: IL-10, IL-22, and tnfβ. Relative mRNA expression levels of IL-10, IL-22, and tnfβ are also shown.

[0134] Figure 18 Section of zebrafish intestinal tissue.

[0135] Figure 19 Detection of recombinant antimicrobial peptides in the fermentation supernatant of engineered bacteria using different signal peptides. From left to right, the bacterial counts of each engineered strain are 1×10⁻⁶. 8 CFU / mL, 2×10 8 CFU / mL, 5×10 8 Western blotting bands of recombinant antimicrobial peptides in fermentation supernatant (unconcentrated) at CFU / mL. Detailed Implementation

[0136] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, and experimental methods for implementing the present invention are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0137] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0138] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0139] This invention discloses a fish-derived antimicrobial peptide, its preparation method, and its applications. The antimicrobial peptide is derived from fish-derived piscidin-1 and hepcidin, and comprises a signal peptide, an antimicrobial peptide, a tag protein, and other functional elements sequentially from the N-terminus to the C-terminus. This invention optimizes the connection method and sequence of piscidin-1, hepcidin, and the signal peptide, antimicrobial peptide, tag protein, and other functional elements in the fish-derived antimicrobial peptide to obtain various engineered bacteria expressing both single-component and tandem fish-derived antimicrobial peptides, achieving optimal bioactivity and functional synergy. The fish-derived antimicrobial peptide and engineered bacteria of this invention can be used as feed additives in fish farming, promoting growth, enhancing immune performance, and improving intestinal flora. This invention has broad application prospects.

[0140] Example 1

[0141] The technical solution of this invention involves constructing an expression vector by adding different signal peptides to the 5' end of the fish-derived piscidin-1 gene (SEQ ID NO:25), transforming it into *Bacillus subtilis* BS168 (Beyotime D0441), and inducing the expression of a soluble product. In vitro, the engineered bacteria can be directly used to detect its antibacterial effect against *Aeromonas hydrophila*; the sfGFP-Piscidin-1 protein is purified to obtain a recombinant antimicrobial peptide product.

[0142] The specific method is as follows:

[0143] I. Construction of the expression vector for the monomeric fish-derived antimicrobial peptide sfGFP-Piscidin-1

[0144] The pisidin-1 mRNA sequence was obtained from NCBI, and the predicted signal peptide sequence was removed, leaving only the antimicrobial peptide sequence. The sequence after removal is as follows.

[0145] Nucleotide sequence of the pisidin-1 antimicrobial peptide gene (SEQ ID NO:25):

[0146] ATGGCTGAACCAGGAGAGTGTTTTGATTGGGACAGTGTTTTAAAAGGAGTCGAAGGCTTTGTCCGGGGGTATTTTGGAAAAGAAAAAGCCAAAGAGTTGGTAAAGTCCCTGAAGGCTGATTTCCAGAATTACAAACATCTGCGTCAACGGGAATTTGACTAG

[0147] The nucleotide sequences of the Bacillus subtilis signal peptides AprE, AmyQ, and SacB were obtained from NCBI and are as follows:

[0148] aprE nucleotide sequence (SEQ ID NO:26):

[0149] ATGAGATCAAAAAAACTGTGGATTTCACTGCGTTTGCACTGACACTGATTTTTACAATGGCA

[0150] amyQ nucleotide sequence (SEQ ID NO:27):

[0151] ATGATTCAAAAACGAAAGCGGACAGTTTCGTTCAGACTTGTGCTTATGTGCACGCTGTTATTTGTCAGTTTGCCGATTACAAAAACATCAGCC

[0152] sacB nucleotide sequence (SEQ ID NO:28):

[0153] ATGAACATCAAGAAATTTGCTAAACAGGCAACGGTCCTGACGTTCACGACCGCACTCTTGGCAGGAGGAGCAACTCAAGCTTTCGCT

[0154] The three signal peptide sequences were linked to the nucleotide sequence of the antimicrobial peptide piscidin-1 gene, and a 6×his tag sequence was added to the 3' end. Codon optimization was performed based on the codon preference of Bacillus subtilis (website: JCat), resulting in three gene fragments named aprE-piscidin-1, amyQ-piscidin-1, and sacB-piscidin-1 (SEQ ID NO: 10-12). These gene fragments were synthesized by Beijing Qingke Biotechnology Co., Ltd., and then ligated into the expression vector pSTOP1622 (Qincheng Biotechnology QCP0276) to obtain plasmids pSTOP1622-aprE-pisidin-1, pSTOP1622-amyQ-pisidin-1, and pSTOP1622-sacB-pisidin-1 (SEQ ID NO: 10-12). Figure 1 ).

[0155] 6×his nucleotide sequence (SEQ ID NO:20):

[0156] CATCACCATCACCATCAC

[0157] Using plasmid pTD103luxI_sfGFP as a template (qincheng BIO QCP2142), the gene fragment sfgfp (SEQ ID NO:29) of the unconventional signal peptide sfGFP was amplified using primers sfGFP-PF / sfGFP-PR (SEQ ID NO:30-31). Its nucleotide sequence is as follows:

[0158] The nucleotide sequence of sfgfp (SEQ ID NO:29):

[0159] Atgagcaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtccgtggagagggtgaaggtgatgctacaaacggaaaa

[0160] ctcacccttaaatttatttgcactactggaaaactacctgttccgtggccaacacttgtcactactctgacctatggtgttcaatgcttttcccgttatccggatcacatgaaacggcatgactttttc

[0161] aagagtgccatgcccgaaggttatgtacaggaacgcactatatctttcaaagatgacgggacctacaagacgcgtgctgaagtcaagtttgaaggtgatacccttgttaatcgtatcgagtta

[0162] aagggtattgattttaaagaagatggaaacattcttggacacaaactcgagtacaactttaactcacacaatgtatacatcacggcagacaaacaaaagaatggaatcaaagctaacttcaaa

[0163] attcgccacaacgttgaagatggttccgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtcgacacaatctgtcctttc

[0164] gaaagatcccaacgaaaagcgtgaccacatggtccttcttgagtttgtaactgctgctgggattacacatggcatggatgagctctacaaa

[0165] The nucleotide sequences of primers sfGFP-P-F / sfGFP-P-R are as follows:

[0166] sfGFP-P-F-atcaaagggggaaatgtacaatgagcaaaggagaagaact (SEQ ID NO:30)

[0167] sfGFP-P-R-TGAACCACCACCACCTGAACCtttgtagagctcatccatgc (SEQ ID NO:31)

[0168] Using plasmid pSTOP1622-aprE-pisidin-1 as a template, the piscidin-1 gene fragment was amplified using primers PF / PR (SEQ ID NO:32-33). Following overlap extension PCR, the sfgfp gene fragment was ligated to the piscidin-1 gene fragment to obtain the target fragment sfgfp-piscidin-1 (SEQ ID NO:16). Next, the expression vector pSTOP1622 was linearized using primers 1622-F / 1622-R (SEQ ID NO:34-35). The target fragment sfgfp-piscidin-1 was integrated into the linearized expression vector pSTOP1622 via homologous recombination to obtain the recombinant plasmid pSTOP1622-sfgfp-piscidin-1 (SEQ ID NO:16). Figure 1 ).

[0169] The nucleotide sequence of primer PF / PR is as follows:

[0170] PF-GGTTCAGGTGGTGGTGGTTCAGCTGAACCTGGCGAA(SEQ ID NO:32)

[0171] PR-cggccggtaccTTAATGATGATGATGATGATGATCGAATTCACG (SEQ ID NO:33)

[0172] The nucleotide sequences of primers 1622-F and 1622-R are as follows:

[0173] 1622-F-ggtaccggccgcatg (SEQ ID NO:34); 1622-R-tgtacatttccccctttgatttaagtgaacaagtttatccaacaac (SEQ ID NO:35) II. Transformation of the monomeric fish-derived antimicrobial peptide sfGFP-Piscidin-1 expression vector

[0174] A single colony of Bacillus subtilis BS168 was streaked onto LB agar and incubated overnight at 37°C. A single colony was then incubated in 10 mL of LB liquid medium at 37°C with shaking overnight (8-10 h). A 2% transfer was made into a 50 mL centrifuge tube containing 8 mL of SPⅠ medium. The culture was incubated at 37°C with shaking until the end of the logarithmic growth phase, approximately 2-3 h, subtracting the blank (OD = 1), at 200 rpm. 0.2 mL (or 2 mL) of the above bacterial suspension was added to 2 mL (or 18 mL) of pre-incubated SPⅡ medium and incubated at 37°C with shaking at 220 rpm for 2 h to obtain competent cells. Immediately, 20 μl of 100X EGTA was added to 2 mL of competent cells and the culture was continued at 37°C for 10 min to obtain the cultured bacterial solution.

[0175] Take 500 μl of the cultured bacterial solution and add it to a preheated 37°C test tube containing pSTOP1622-aprE-pisidin-1, pSTOP1622-amyQ-pisidin-1, pSTOP1622-sacB-pisidin-1, and pSTOP1622-sfgfp-piscidin-1 plasmid DNA (no more than 2% of the bacterial solution, i.e., 5-10 μl). Incubate at 37°C with shaking at 200 rpm for 2 hours. (If the cells lyse and the solution becomes clear, the plasmid DNA obtained in step one may contain chloroform and phenol, requiring dialysis or re-precipitation of the DNA with ethanol). Add 100 μl of LB liquid medium and continue incubation at 37°C with shaking for 1 hour. Centrifuge at 10,000 rpm for 1 minute to remove the supernatant, obtaining a bacterial pellet. Resuspend the bacterial pellet in 200 μl of LB liquid medium, spread the suspension on a selective plate, and incubate overnight at 37°C. Single clones were selected and sequenced to identify the transformation results, yielding engineered bacteria BS168-AprE-Pisidin-1, BS168-AmyQ-Pisidin-1, BS168-SacB-Pisidin-1, and BS168-sfGFP-Pisidin-1.

[0176] The LB solid medium formula is as follows: 1% tryptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, and ddH2O.

[0177] The LB liquid culture medium is formulated as follows: 1% tryptone, 0.5% yeast extract, 1% NaCl, and ddH2O.

[0178] The formulation of SPⅠ medium is as follows: 0.2% (NH4)2SO4, 1.4% K2HPO4, 0.6% KH2PO4, 0.1% NaCitrate·2H2O, 0.02% MgSO4·7H2O, 1% glucose, and 1% CAYE. The formulation of SPⅡ medium is as follows: SPI, 1% CaCl2, and 1% MgCl2.

[0179] III. Functional Verification of Engineered Microorganisms

[0180] The engineered bacteria BS168-AprE-Pisidin-1, BS168-AmyQ-Pisidin-1, BS168-SacB-Pisidin-1, and BS168-sfGFP-Pisidin-1 obtained in step two were induced for 24 hours in LB liquid medium with 1.5% xylose. The bacterial culture was then diluted to 10. 8 CFU / mL.

[0181] Single colonies of Aeromonas hydrophila were inoculated into fresh LB broth and incubated at 37°C and 200 rpm for 12 hours. The bacterial culture was then diluted to 10⁻⁶. 7 CFU / mL. Add 100 μL of bacterial suspension to LB solid medium. After the medium has completely solidified, punch holes in the medium using a 9 mm diameter punch and add 200 μL of diluted bacterial suspension of each engineered strain to the holes. Incubate the plates at 37°C for 12 h and measure the diameter of the inhibition zone.

[0182] Experimental results showed that all engineered bacteria had inhibitory effects on Aeromonas hydrophila, among which the engineered bacteria BS168-sfGFP-Pisidin-1, which used sfGFP as the signal peptide, had the best antibacterial effect. Figure 2 )

[0183] The formulation of LB liquid medium is the same as that of LB liquid medium in step one; the formulation of LB solid medium is the same as that of LB solid medium in step one. IV. Purification of recombinant fish-derived antimicrobial peptide protein sfGFP-Piscidin-1 (SEQ ID NO:4)

[0184] The engineered strain BS168-sfGFP-Pisidin-1 obtained in step two was induced in LB liquid medium with 1.5% xylose for 24 h. The cells were collected by centrifugation, the supernatant was discarded, and the cells were resuspended in pre-cooled lysis buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH = pI-2) and transferred to 50 mL centrifuge tubes. The cells were then sonicated. Centrifuged at 12000 rpm for 20 minutes at 4 °C, and the supernatant and precipitate were collected separately to determine the solubility of the target protein. Equilibrate the nickel column with 5 times its volume (referring to the packing material volume) of protein washing buffer (20 mM Tris, 500 mM NaCl, pH = pI ± 2, pH stability range of 5-9 for 3.5 kDa dialysis bags); load the supernatant after sonication and centrifugation onto the column at a flow rate of 4 s / drop to ensure sufficient binding of the target protein to the nickel column, and collect the permeate; wash the nickel column with 4 times its volume of pre-cooled protein washing buffer to remove non-specifically bound proteins, and collect the washing buffer; elute the target protein with 4 times its volume of pre-cooled protein elution buffer (20 mM Tris, 500 mM NaCl, imidazole concentration gradient of 30-500 mM, pH = pI ± 2), and collect the elution buffer; perform SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining on a small amount of sample, and store the remaining samples at 4℃; based on the staining results, select the elution buffer containing the purest target protein and dialyze at 4℃ (dialysis buffer: 20 mM Tris, 500 mM NaCl, pH = pI ± 2, pH = pI ± 2). Tris (pH = pI⁻²), the dialysis buffer was changed every 4 hours, and the dialysis results could be monitored using a conductivity meter. The dialyzed protein solution was concentrated by ultrafiltration, centrifuged at 4000g at 4°C to 3-5 mL, and a small amount was taken to determine protein purity and concentration. The remaining sample was sterilized by filtration in a sterile laminar flow hood, aliquoted, and stored at -80°C for later use. The final concentration of the ultrafiltered protein sfGFP-Piscidin-1 was 183 μg / mL. Figure 3 )

[0185] The formulation of LB liquid medium is the same as that of LB liquid medium in step one.

[0186] V. In vitro antibacterial activity of recombinant fish-derived antimicrobial peptide protein sfGFP-Piscidin-1 (SEQ ID NO:4)

[0187] Single colonies of Aeromonas hydrophila were inoculated onto fresh LB liquid medium and incubated at 37°C and 200 rpm until OD500 was reached. 600 The concentration was 0.5. 20 μM sfGFP-Piscidin-1 and an equal volume of PBS (negative control) were added to the Aeromonas hydrophila bacterial culture, and 100 ng / mL ampicillin (AMP+) was set as a positive control. The mixture was incubated in a shaker at 37°C, and the OD of each group was measured every 3 hours. 600 .

[0188] Experimental results showed that the recombinant fish-derived antimicrobial peptide protein sfGFP-Piscidin-1 could effectively inhibit the growth of Aeromonas hydrophila, and this inhibitory effect was similar to that of antibiotics. Figure 4 )

[0189] The formulation of LB liquid medium is the same as that of LB liquid medium in step one.

[0190] Example 2

[0191] The technical solution of this invention involves constructing an expression vector by adding different signal peptides to the 5' end of the fish-derived hepcidin gene (SEQ ID NO:36), transforming it into *Bacillus subtilis* BS168 (Beyotime D0441), and inducing the expression of a soluble product. In vitro, the engineered bacteria can be directly used to detect its antibacterial effect against *Aeromonas hydrophila*; the sfGFP-Hepcidin protein is purified to obtain a recombinant antimicrobial peptide product.

[0192] The specific method is similar to the construction, transformation, engineered bacterial function verification, protein purification, and antibacterial activity verification of the recombinant antimicrobial peptide sfGFP-Piscidin-1 expression vector in Example 1 of this invention. Specifically, the Hepcidin mRNA sequence was obtained from NCBI, the predicted signal peptide sequence was removed, and only the antimicrobial peptide sequence was retained. The sequence after removal is as follows:

[0193] Nucleotide sequence of the hepcidin antimicrobial peptide gene (SEQ ID NO:36)

[0194] ATCCCATTTGCTGGGGTGCAAGAGCCGGAGGAGGCAGGGAGCAATGACACTCCAGTTGTGGCACATCAAGAGATG

[0195] TCAGCGGAGTCATCAATGATGTCAAATCACATCAGGCAAAAGCGTCAGAGCCATCTTTCCTTGTGCCGCTGGTGCT

[0196] GCAATTGCTGCCGAAGCAACAAGGGCTGCGGCTTCTGCTGCAGGTTCTGA

[0197] The three signal peptide sequences were linked to the nucleotide sequences of the antimicrobial peptide hepcidin gene, and a 6×his tag sequence (SEQ ID NO:20) was added to the 3' end. Codon optimization was performed based on the codon preference of Bacillus subtilis (website: JCat), resulting in three gene fragments named aprE-hepcidin, amyQ-hepcidin, and sacB-hepcidin (SEQ ID NO:13-15), respectively. These gene fragments were synthesized by Beijing Qingke Biotechnology Co., Ltd., and then ligated into the expression vector pSTOP1622 (Qincheng Biotechnology QCP0276) to obtain plasmids pSTOP1622-aprE-hepcidin, pSTOP1622-amyQ-hepcidin, and pSTOP1622-sacB-hepcidin. Figure 5 ).

[0198] Using plasmid pSTOP1622-aprE-hepcidin as a template, the hepcidin gene fragment was amplified using primers HF / HR (SEQ ID NO:37-38). Following overlap extension PCR, sfgfp (SEQ ID NO:29) was ligated to hepcidin to obtain the target fragment sfgfp-hepcidin (SEQ ID NO:17). Next, the expression vector pSTOP1622 was linearized using primers 1622-F / 1622-R (SEQ ID NO:34-35). The target fragment sfgfp-hepcidin was integrated into the linear expression vector pSTOP1622 via homologous recombination to obtain the recombinant plasmid pSTOP1622-sfgfp-hepcidin (SEQ ID NO:17). Figure 5 ).

[0199] The nucleotide sequence of primer HF / HR is as follows:

[0200] HF-GGTTCAGTGGTGGTGGTTCAATCCCTTTCGCTGGC (SEQ ID NO:37)

[0201] HR-gccggcatgcggccggtacctgattggctccaattcttgga (SEQ ID NO:38)

[0202] The above plasmids were transformed into BS168 using the same transformation method as in Part 2 of Example 1 of this invention, resulting in engineered bacteria BS168-AprE-Hepcidin, BS168-AmyQ-Hepcidin, BS168-SacB-Hepcidin, and BS168-sfGFP-Hepcidin.

[0203] The verification method was the same as that in the third part of Example 1 of this invention. The engineered bacteria using AprE and sfGFP as signal peptides showed inhibitory effects against Aeromonas hydrophila, with the engineered bacteria BS168-sfGFP-Hepcidin exhibiting the best antibacterial effect. Figure 6 )

[0204] The recombinant antimicrobial peptide protein sfGFP-Hepcidin (SEQ ID NO:8) was purified using the same purification method as in Part IV of Example 1 of this invention, yielding a final concentration of 107 μg / mL for sfGFP-Hepcidin. Figure 7 ).

[0205] As verified, using the same verification method as in Part 5 of Embodiment 1 of this invention, the recombinant antimicrobial peptide protein sfGFP-Hepcidin can effectively inhibit the growth of Aeromonas hydrophila. Figure 8 )

[0206] Example 3

[0207] The technical solution of this invention involves constructing a tandem fish-derived piscidin-1 gene (SEQ ID NO:25) and a fish-derived hepcidin gene (SEQ ID NO:36), and transforming *Bacillus subtilis* BS168 (Beyotime D0441) using an expression vector loaded with the atypical signal peptide sfGFP gene, to induce the expression of a soluble product. *Bacillus subtilis* can fold heterologous proteins to a certain extent when expressing them, which makes most of the heterologous proteins expressed by *Bacillus subtilis* possess certain activities. Furthermore, it can undergo secretory expression, providing a good fermentation basis. Simultaneously, as a probiotic, it also has the advantages of improving animal intestinal function and enhancing animal immunity. In vitro, the sfGFP-PH-his protein is purified to obtain the tandem expression product; in vivo, by adding xylose, which is harmless to fish, to the feed, the engineered bacteria are induced to express tandem antimicrobial peptides in the fish intestine, improving the fish's disease resistance and survival rate.

[0208] The specific method is as follows:

[0209] I. Construction of the Expression Carrier

[0210] The mRNA sequences of pisidin-1 and hepcidin were obtained from NCBI. The signal peptide sequence was predicted and then removed, leaving only the antimicrobial peptide sequence. The sequences after removal are shown in SEQ ID NO:25 and 36.

[0211] The gene sequences of the two antimicrobial peptides mentioned above were linked together by the enterokinase site sequence, and a 6×His tag was added to the C-terminus. Codon optimization was performed based on the codon preference of Bacillus subtilis (website: JCat) to obtain the gene fragment, named ph. This fragment was synthesized by Beijing Qingke Biotechnology Co., Ltd., and then ligated into the cloning vector pUC57 by the aforementioned company, resulting in the cloning vector pUC57-ph.

[0212] Nucleotide sequence of the enterokinase site (DDDDK (SEQ ID NO:23)): GATGATGATAAA (SEQ ID NO:24)

[0213] The cloning vector pUC57-PH containing the PH fragment was extracted and amplified using primers PH-F and PH-R (SEQ ID NO:39-40). sfGFP was then added to the N-terminus of the target fragment sfgfp-ph via overlap extension PCR to obtain the target fragment sfgfp-ph. Linkerg4s (SEQ ID NO:22) was designed into the PCR primers and added between sfgfp and ph. Next, the expression vector pSTOP1622 was linearized using primers 1622-F / 1622-R (SEQ ID NO:34-35). The aforementioned target fragment sfGFP-PH was integrated into the linearized expression vector pSTOP1622 via homologous recombination to obtain the recombinant plasmid pSTOP1622-sfgfp-ph-his( Figure 9 Sequencing results showed that the target fragment was successfully inserted into the expression vector and read correctly. Amplification was performed upstream and downstream of the target fragment using universal primers SPST-F and SPST-R, yielding a fragment of 1501 bp. Its agarose gel electrophoresis image is shown below. Figure 10 As shown, con: Bacillus subtilis BS168 transformed with empty vector pSTOP1622.

[0214] The nucleotide sequences of primers PH-F and PH-R are as follows:

[0215] PH-F-GGTTCAGGTGGTGGTGGTTCAGCTGAACCTGGCGAA(SEQ ID NO:39)

[0216] PH-R-gccggcatgcggccggtacctgattggctccaattcttgga(SEQ ID NO:40)

[0217] The nucleotide sequences of primers SPST-F and SPST-R are as follows:

[0218] SPST-F-cctttgtttatccaccgaact (SEQ ID NO:41); SPST-R-cgcaagaattgattggctcc (SEQ ID NO:42)

[0219] II. Preparation of Tandem Expression Products

[0220] (1) Expression vector transformed into Bacillus subtilis BS168

[0221] The recombinant plasmid pSTOP1622-sfgfp-ph-his obtained in step one was transformed into BS168 using the same transformation method as in the second part of Example 1 of the present invention, to obtain BS168-sfGFP-PH-his.

[0222] (2) Verification of the expression of recombinant fish-derived antimicrobial peptides

[0223] The engineered strain BS168-sfGFP-PH-his obtained in step (1) was induced with 1.5% xylose in LB liquid medium. Under blue light, obvious green fluorescence was observed in the engineered strain BS168-sfGFP-PH-his, proving the expression of the target gene. Figure 11 )

[0224] The formulation of the LB liquid culture medium is the same as that of the LB liquid culture medium described in Example 1 of this invention.

[0225] (3) Nickel column purification of recombinant fish-derived antimicrobial peptides

[0226] The purification method was the same as in Part IV of Example 1 of this invention. The ultrafiltered recombinant fish-derived antimicrobial peptide protein sfGFP-PH-his (SEQ ID NO:9) showed a single band in SDS-PAGE, with a final concentration of 156 μg / mL. Figure 12 )

[0227] III. In vivo induction methods and biological functions of engineered bacteria

[0228] Uninduced engineered bacteria BS168-sfGFP-PH-his were added to zebrafish feed (10). 8 (CFU / g) was added, along with 1% xylose for in vivo induction. Zebrafish fed with the engineered bacteria induced in vitro for 12 hours and those without the engineered bacteria were used as controls. Six hours after feeding, the zebrafish intestines were collected and observed under a fluorescence microscope. Figure 5 As shown, fluorescence microscopy revealed obvious green fluorescence in the intestines of zebrafish induced in vivo, proving that the engineered bacterium BS168-sfGFP-PH-his can achieve in vivo induction. Figure 13 )

[0229] Zebrafish are fed commercial feed (Shandong Shengsuo Feed Technology Co., Ltd.)

[0230] Main ingredients: imported specialty fish meal, Antarctic krill meal, refined fish oil, lecithin, taurine, choline chloride, vitamins and vitamin-like substances, and complex minerals.

[0231] Add 10g to the zebrafish feed. 8 Uninduced engineered bacteria BS168-sfGFP-Piscidin-1 (prepared in Example 1 of this invention), BS168-sfGFP-Hepcidin (prepared in Example 2 of this invention), and BS168-sfGFP-PH-his (prepared in this example) at CFU / g were fed for three days and then challenged with Aeromonas hydrophila by immersion (10). 7 (CFU / mL). Simultaneously, the addition of 1% xylose to the feed induced the expression of recombinant fish-derived antimicrobial peptides sfGFP-Piscidin-1 (SEQ ID NO:4), sfGFP-Hepcidin (SEQ ID NO:8), and sfGFP-PH-his (SEQ ID NO:9). The following experiments were conducted:

[0232] 1. For zebrafish fed as described above, the survival rate of zebrafish was statistically analyzed. Compared with the control group (BS168-con, i.e., BS168 transformed with empty vector plasmid pSTOP1622), feeding the engineered bacteria BS168-sfGFP-PH-his and inducing the expression of the fish-derived antimicrobial peptide sfGFP-PH-his in vivo can effectively save the survival rate of zebrafish. Figure 14 )

[0233] 2. For zebrafish fed as described above, using the Aerolysin gene of Aeromonas hydrophila as the target gene, specific primers AHaerA-F and AHaerA-R (SEQ ID NO: 43-44) were designed. The specific fragment was amplified by PCR and ligated into the vector pMD19-T (TaKaRa code 6013pMD19-T Vector Cloning Kit) to obtain the plasmid pMD19-T-AHaerA. A standard curve was constructed using the pMD19-T-AHaerA plasmid DNA as a template, and the gene copy number of Aeromonas hydrophila in the zebrafish intestinal contents was calculated. Compared with the control group (BS168-con, i.e., BS168 transformed with the empty vector plasmid pSTOP1622), feeding with the engineered bacteria significantly reduced the level of Aeromonas hydrophila in the zebrafish intestine. Figure 15 )

[0234] The nucleotide sequences of primers AHaerA-F and AHaerA-R are as follows:

[0235] AHaerA-F-GCTCCAAGATCCCGGTGAAG (SEQ ID NO: 43); AHaerA-R-GCGGTTGTCCGGATGGGTAT (SEQ ID NO: 44)

[0236] 3. RNA was extracted from the zebrafish intestinal tissue of the zebrafish fed as described above and reverse transcribed into cDNA. Using the cDNA as a template, the expression levels of inflammatory factors (including pro-inflammatory factors TNFA, IL-1β, CXCL8, and IL-6; and anti-inflammatory factors IL-10, IL-22, and TNFβ) in the zebrafish intestinal tissue were detected by qPCR. This reduced the expression levels of pro-inflammatory factors in the zebrafish intestine. Figure 16 This increased the expression level of anti-inflammatory factors. Figure 17 ).

[0237] 4. Furthermore, in zebrafish fed as described above, after the zebrafish intestines were fixed, dehydrated, embedded, sectioned, and stained, observation under an optical microscope showed that feeding engineered bacteria increased the thickness of the lamina propria of the zebrafish intestines, playing an important role in maintaining intestinal homeostasis. Figure 18 )

[0238] The results of absolute quantification of Aeromonas hydrophila in the intestine are as follows: Figure 15 As shown. The results of qPCR detection of pro-inflammatory factors are as follows. Figure 16 As shown. The results of qPCR detection of anti-inflammatory factors are as follows. Figure 17 As shown. The results of H&E staining of intestinal sections are as follows. Figure 18 As shown.

[0239] The primers used in qPCR are as follows:

[0240] Tnf-aF-TCACGCTCCATAAGACCCAG (SEQ ID NO: 45); Tnf-aR-AAATGGATGGCAGCCTTGGA (SEQ ID NO: 46) il-1b-F-ACGTCATCCAAGAGCGTGAA (SEQ ID NO: 47); il-1b-R-CCTGCAAATGTGGAGATCCG (SEQ ID NO: 48) Cxcl8-F-AAGCCGACGCATTGGAAAAC (SEQ ID NO: 49); Cxcl8-R-GTTGTCATCAAGGTGGCAATGA (SEQ ID NO: 50) il-6-F-AGCTTCTGGACACATATAAAGCCA (SEQ ID NO: 51); il-6-R-AAACGCCTTCAGGTGGGAAT (SEQ ID NO:52)il-10-F-GCTCTGCTCACGCTTCTTCT (SEQ ID NO: 53); il-10-R-CCAAGTCATCGTTGGATTCATAAAA (SEQ ID NO: 54) il-22-F-CACGACTACTCCAGCCATCC (SEQ ID NO: 55); il-22-R-GGCTGCGGCCAAATCCATAA (SEQ ID NO: 56) Tgfb-F-TCCTCTTCACTATATCAGGGCT (SEQ ID NO:57); Tgfb-R-GTGCCAACAGCTCGTCTCT (SEQ ID NO:58)

[0241] Compared with existing research reports, the features of this invention are:

[0242] (1) Non-constitutive expression of antimicrobial peptides has achieved the separation of the growth period and expression period of engineered bacteria, and has minimized the impact of secreted antimicrobial peptides on the chassis strain itself.

[0243] (2) In vivo induction of expression enabled precise regulation of the protective effect of engineered bacteria on fish.

[0244] (3) Visualize the induced expression process using sfGFP as a signal peptide molecule;

[0245] (4) Using Bacillus subtilis as a substrate is beneficial to achieving the superposition of the probiotic effect of Bacillus subtilis and the antibacterial effect of recombinant fish-derived antimicrobial peptides;

[0246] (5) Feeding the engineered bacteria BS168-sfGFP-PH-his and inducing it in vivo effectively improved the survival rate of zebrafish challenged with Aeromonas hydrophila and improved their immune performance.

[0247] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0248] As used in this invention, the terms "comprising" and "including" are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0249] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0250] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of this invention are included in this invention and are protected by the appended claims.

[0251] In this invention, the sequence information is as follows:

[0252] AprE-Pisidin-1 protein sequence (SEQ ID NO:1)

[0253] MRSKKLWISLLFALTLIFTMAMAEPGECFDWDSVLKGVEGFVRGYFGKEKAKELVKSLKADFQNYKHLRQREFDHHHHHHH*

[0254] AmyQ-Pisidin-1 protein sequence (SEQ ID NO:2)

[0255] MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAAEPGECFDWDSVLKGVEGFVRGYFGKEKAKELVKSLKADFQNYKHLRQREFDHHHHHHH*

[0256] SacB-Pisidin-1 protein sequence (SEQ ID NO:3)

[0257] MNIKKFAKQATVLTFTTALLAGGATQAFAAEPGECFDWDSVLKGVEGFVRGYFGKEKAKELVKSLKADFQNYKHLRQREFDHHHHHHH*

[0258] sfGFP-Piscidin-1 Protein Sequence (SEQ ID NO:4)

[0259] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGGGGSAEPGECFDWDSVLKGVEGFVRGYFGKEKAKELVKSLKADFQNYKHLRQREFDHHHHHH*

[0260] AprE-Hepcidin Protein Sequence (SEQ ID NO:5)

[0261] MRSKKLWISLLFALTLIFTMAIPFAGVQEPEEAGSNDTPVVAHQEMSAESSMMSNHIRQKRQSHLSLCRWCCNCCRSNKGCGFCCRFHHHHHH*

[0262] AmyQ-Hepcidin Protein Sequence (SEQ ID NO:6)

[0263] MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAIPFAGVQEPEEAGSNDTPVVAHQEMSAESSMMSNHIRQKRQSHLSLCRWCCNCCRSNKGCGFCCRFHHHHHH*

[0264] SacB-Hepcidin Protein Sequence (SEQ ID NO:7)

[0265] MNIKKFAKQATVLTFTTALLAGGATQAFAIPFAGVQEPEEAGSNDTPVVAHQEMSAESSMMSNHIRQKRQSHLSLCRWCCNCCRSNKGCGFCCRFHHHHHH*

[0266] sfGFP-Hepcidin Protein Sequence (SEQ ID NO:8)

[0267] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGGGGSIPFAGVQEPEEAGSNDTPVVAHQEMSAESSMMSNHIRQKRQSHLSLCRWCCNCCRSNKGCGFCCRFHHHHHH*

[0268] sfGFP-PH-his protein sequence (SEQ ID NO:9)

[0269] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYKGSGGGGSAEPGECFDWDSVLKGVEGFVRGYFGKEKAKELVKSLKADFQNYKHLRQREFDDDDDKGSGGGGSIPFAGVQEPEEAGSNDTPVVAHQEMSAESSMMSNHIRQKRQSHLSLCRWCCNCCRSNKGCGFCCRFHHHHHH*

[0270] aprE-piscidin-1 nucleotide sequence (SEQ ID NO:10)

[0271] ATGAGATCAAAAAAACTGTGGATTTCACTGCTGTTTGCACTGACACTGATTTTTACAATGGCAATGGCTGAACCTGGCGAATGCTTCGATTGGGATTCTGTTCTTAAAGGCGTTGAAGGCTTCGTTCGTGGCTACTTCGGCAAAGAAAAAGCTAAAGAACTTGTTAAATCTCTTAAAGCTGATTTCCAAAACTACAAACATCTTCGTCAACGTGAATTCGATTAA

[0272] Nucleotide sequence of amyQ-piscidin-1 (SEQ ID NO:11)

[0273] ATGATTCAAAAACGAAAGCGGACAGTTTCGTTCAGACTTGTGCTTATGTGCACGCTGTTATTTGTCAGTTTGCCGATTACAAAAACATCAGCCGCTGAACCTGGCGAATGCTTCGATTGGGATTCTGTTCTTAAAGGCGTTGAAGGCTTCGTTCGTGGCTACTTCGGCAAAGAAAAAGCTAAAGAACTTGTTAAATCTCTTAAAGCTGATTTCCAAAACTACAAACATCTTCGTCAACGTGAATTCGATCATCACCATCACCATCACTAA

[0274] Nucleotide sequence of sacB-piscidin-1 (SEQ ID NO:12)

[0275] ATGAACATCAAGAAATTTGCTAAACAGGCAACGGTCCTGACGTTCACGACCGCACTCTTGGCAGGAGGAGCAACTCAAGCTTTCGCTGCTGAACCTGGCGAATGCTTCGATTGGGATTCTGTTCTTAAAGGCGTTGAAGGCTTCGTTCGTGGCTACTTCGGCAAAGAAAAAGCTAAAGAACTTGTTAAATCTCTTAAAGCTGATTTCCAAAACTACAAACATCTTCGTCAACGTGAATTCGATTAA

[0276] Nucleotide sequence of aprE-hepcidin (SEQ ID NO:13)

[0277] ATGAGATCAAAAAAACTGTGGATTTCACTGCTGTTTGCACTGACACTGATTTTTACAATGGCAATCCCTTTCGCTGGCGTTCAAGAACCTGAAGAAGCTGGCTCTAACGATACACCTGTTGTTGCTCATCAAGAAATGTCTGCTGAATCTTCTATGATGTCTAACCATATCCGTCAAAAACGTCAATCTCATCTTTCTCTTTGCCGTTGGTGCTGCAACTGCTGCCGTTCTAACAAAGGCTGTGGCTTCTGCTGCAGGTTCCATCATCATCATCATCATTGA

[0278] amyQ-hepcidin nucleotide sequence (SEQ ID NO:14)

[0279] ATGATTCAAAAACGAAAGCGGACAGTTTCGTTCAGACTTGTGCTTATGTGCACGCTGTTATTTGTCAGTTTGCCGATTACAAAAACATCAGCCATCCCTTTCGCTGGCGTTCAAGAACCTGAAGAAGCTGGCTCTAACGATACACCTGTTGTTGCTCATCAAGAAATGTCTGCTGAATCTTCTATGATGTCTAACCATATCCGTCAAAAACGTCAATCTCATCTTTCTCTTTGCCGTTGGTGCTGCAACTGCTGCCGTTCTAACAAAGGCTGTGGCTTCTGCTGCAGGTTCCATCATCATCATCATCATTGA

[0280] sacB-hepcidin nucleotide sequence (SEQ ID NO:15)

[0281] ATGAACATCAAGAAATTTGCTAAACAGGCAACGGTCCTGACGTTCACGACCGCACTCTTGGCAGGAGGAGCAACTCAAGCTTTCGCTATCCCTTTCGCTGGCGTTCAAGAACCTGAAGAAGCTGGCTCTAACGATACACCTGTTGTTGCTCATCAAGAAATGTCTGCTGAATCTTCTATGATGTCTAACCATATCCGTCAAAAACGTCAATCTCATCTTTCTCTTTGCCGTTGGTGCTGCAACTGCTGCCGTTCTAACAAAGGCTGTGGCTTCTGCTGCAGGTTCCATCATCATCATCATCATTGA

[0282] Nucleotide sequence of sfgfp-piscidin-1 (SEQ ID NO:16)

[0283] atgagcaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtccgtggagagggtgaaggtgatgctacaaacggaaaactcacccttaaatttatttgcactactggaaaactacctgttccgtggccaacacttgtcactactctgacctatggtgttcaatgcttttcccgttatccggatcacatgaaacggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaacgcactatatctttcaaagatgacgggacctacaagacgcgtgctgaagtcaagtttgaaggtgatacccttgttaatcgtatcgagttaaagggtattgattttaaagaagatggaaacattcttggacacaaactcgagtacaactttaactcacacaatgtatacatcacggcagacaaacaaaagaatggaatcaaagctaacttcaaaattcgccacaacgttgaagatggttccgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaaccattacctgtcgacacaatctgtcctttcgaaagatcccaacgaaaagcgtgaccacatggtccttcttgagtttgtaactgctgctgggattacacatggcatggatgagctctacaaaGGTTCAGGTGGTGGTGGTTCAGCTGAACCTGGCGAATGCTTCGATTGGGATTCTGTTCTTAAAGGCGTTGAAGGCTTCGTTCGTGGCTACTTCGGCAAAGAAAAAGCTAAAGAACTTGTTAAATCTCTTAAAGCTGATTTCCAAAACTACAAACATCTTCGTCAACGTGAATTCGATCATCATCATCATCATCATTAA

[0284] Nucleotide sequence of sfgfp-hepcidin (SEQ ID NO:17)

[0285] atgagcaaaggagaagaacttttcactggagttgtcccaattcttgttgaattagatggtgatgttaatgggcacaaattttctgtccgtggagagggtgaaggtgatgctacaaacggaaaactcacccttaaatttatttgcactactggaaaactacctgttccgtggccaacacttgtcactactctgacctatggtgttcaatgcttttcccgttatccggatcacatgaaac ggcatgactttttcaagagtgccatgcccgaaggttatgtacaggaacgcactatatctttcaaagatgacgggacctacaagacgcgtgctgaagtcaagtttgaaggtgatacccttgttaatcgtatcgagttaaagggtattgattttaaagaagatggaaacattcttggacacaaactcgagtacaactttaactcacacaatgtatacatcacggcagacaaacaaaagaat ggaatcaaagctaacttcaaaattcgccacaacgttgaagatggttccgttcaactagcagaccattatcaacaaaatactccaattggcgatggccctgtccttttaccagacaacca ttacctgtcgacacaatctgtcctttcgaaagatcccaacgaaaagcgtgaccacatggtccttcttgagtttgtaactgctgctgggattacacatggcatggatgagctctacaaag gttcaGGTGGTGGTGGTTCAATCCCTTTCGCTGGCGTTCAAGAACCTGAAGAAGCTGGCTCTAACGATACACCTGTTGTTGCTCATCAAGAAATGTCTGCTGAATCTTCTATGATGTCTAACCATATCCGTCAAAAACGTCAATCTCATCTTTCTCTTTGCCGTTGGTGCTGCAACTGCTGCCGTTCTAACAAAGGCTGTGGCTTCTGCTGCAGGTTCCATCATCATCATCATCATTGA

[0286] sfgfp-ph-his nucleotide sequence (SEQ ID NO:18)

[0287]

[0288] 6×His amino acid sequence (SEQ ID NO:19)

[0289] HHHHHH

[0290] 6×his nucleotide sequence (SEQ ID NO:20)

[0291] CATCACCATCACCATCAC

[0292] G4S amino acid sequence (SEQ ID NO:21)

[0293] GSGGGGS

[0294] Linker g4S nucleotide sequence (SEQ ID NO:22)

[0295] GGTTCAGGTGGTGGTGGTTCA

[0296] Amino acid sequence of the enterokinase site (SEQ ID NO:23)

[0297] DDDDK

[0298] Nucleotide sequence of the enterokinase site (SEQ ID NO:24)

[0299] GATGATGATGATAAA

[0300] References:

[0301] [1]GRZIA MTS, CARMELO C, PAOLO A, et al. Circular economy in the foodchain: Retrieval and characterization of antimicrobial peptides from fishwaste hydrolysates[J]. Food Analytical Methods, 2023, 17(2): 178-199.

[0302] [2]ZHANG

[0303] [3]GUAN Feng, LI Chang-Hong, NIE Li, MIAO Liang, CHEN Jiong.2018.PISCIDIN1OF MUDSKIPPER BOLEOPHTHALMUS PECTINIROSTRIS:MOLECULAR CHARACTERIZATION ANDFUNCTIONALANALYSIS[J].Oceanologia etLimnologia Sinica,49(2):422-431.

[0304] [4] Cervera L, Arizcun M, Mercado L, Cuesta A, Chaves-Pozo E. Hepcidinpeptide controls the inflammatory response induced by betatanodavirus infection and improves European seabass (Dicentrachus labrax) survival. Mar Life SciTechnol. 2025Jan 23;7(1):110-119.doi:10.1007 / s42995-024-00262-w.PMID:40027333; PMCID:PMC11871212.

[0305] [5] Huang Jin, Huang Junyan, Ke Tao, et al. Cloning, expression and activity detection of antimicrobial peptide gene BnPCD842895 [J]. Chinese Journal of Oil Crop Sciences, 2013, 35(04):357-363.

[0306] [6]Zhang Z,Li Y,Zheng L,et al.A novel method forhigh level productionofprotein glutaminase by sfGFP tag in Bacillus subtilis[J].InternationalJournal ofBiological Macromolecules,2024,262.

Claims

1. A fish-derived antibacterial peptide, characterized in that, The fish-derived antibacterial peptide comprises, from N-terminal to C-terminal, a signal peptide, an antibacterial peptide, and a tag protein.

2. The fish-derived antibacterial peptide according to claim 1, wherein The fish-derived antibacterial peptide comprises a recombinant antibacterial peptide; and / or, the fish-derived antibacterial peptide comprises one or both of a monomeric fish-derived antibacterial peptide and a tandem fish-derived antibacterial peptide; and / or, a linker is further comprised between the signal peptide and the antibacterial peptide; and / or, the signal peptide comprises one or more of a Bacillus subtilis signal peptide AprE, AmyQ, SacB, and an unconventional signal peptide sfGFP; and / or, the antibacterial peptide comprises one or both of a fish-derived Piscidin-1 and a fish-derived Hepcidin; and / or, the tag protein comprises 6xHis; and / or, when the fish-derived antibacterial peptide is formed by tandem of the fish-derived Piscidin-1 and / or the fish-derived Hepcidin, an enterokinase site sequence is further comprised between individual antibacterial peptides.

3. A polynucleotide, comprising, The polynucleotide comprises a polynucleotide encoding the fish-derived antibacterial peptide according to claim 1 or 2.

4. A substance characterized in that, The substance comprises one or more of: (1) a recombinant vector comprising the polynucleotide according to claim 3; (2) a transformant comprising one or both of the polynucleotide according to claim 3 and the recombinant vector according to (1); (3) a drug / medicinal composition comprising one or more of the polynucleotide according to claim 3, the recombinant vector according to (1), and the transformant according to (2); (4) a feed additive comprising one or more of the polynucleotide according to claim 3, the recombinant vector according to (1), and the transformant according to (2); (5) a probiotic preparation comprising the transformant according to (2); (6) a postbiotic comprising the fish-derived antibacterial peptide according to claim 1; (7) a primer comprising one or more of sfGFP-P-F, sfGFP-P-R, P-F, P-R, 1622-F, 1622-R, H-F, H-R, PH-F, PH-R, SPST-F, SPST-R, AHaerA-F, AHaerA-R, Tnf-a-F, Tnf-a-R, il-1b-F, il-1b-R, Cxcl8-F, Cxcl8-R, il-6-F, il-6-R, il-10-F, il-10-R, il-22-F, il-22-R, Tgfb-F, and Tgfb-R. (8) a primer pair comprising one or more of sfGFP-P-F / sfGFP-P-R, P-F / P-R, 1622-F / 1622-R, H-F / H-R, PH-F / PH-R, SPST-F / SPST-R, AHaerA-F / AHaerA-R, Tnf-a-F / Tnf-a-R, il-1b-F / il-1b-R, Cxcl8-F / Cxcl8-R, il-6-F / il-6-R, il-10-F / il-10-R, il-22-F / il-22-R, Tgfb-F / Tgfb-R; (9) a nucleotide comprising one or both of enterokinase site sequence, linker g4s; (10) a polypeptide comprising one or both of enterokinase site, G4S.

5. A method characterized by, The method comprises one or more of the following: (1) a preparation method of the fish-derived antibacterial peptide according to claim 1, comprising the following steps: Step 1: constructing the recombinant vector in (1) according to claim 4; Step 2: transforming the recombinant vector constructed in Step 1 into a host to obtain a transformant; Step 3: expressing the transformant obtained in Step 2 to obtain an expression product, i.e. the fish-derived antibacterial peptide; (2) an in vivo induction method of the transformant according to claim 4 in (2), comprising adding the transformant to animal feed and adding an inducer for in vivo induction; (3) a fermentation and concentration method of the probiotic preparation according to claim 4 in (5), comprising preparing a seed, expanding culture, fermentation culture, and washing and concentrating.

6. The method of claim 5, wherein, In (1), the preparation method further comprises verifying the fish-derived antibacterial peptide obtained in Step 3; and / or, the preparation method further comprises purifying the fish-derived antibacterial peptide obtained in Step 3; and / or, In (2), the animal comprises a farmed animal; and / or, the recombinant vector contained in the transformant comprises a recombinant vector without a resistance gene tag; and / or, In (3), the fermentation and concentration method comprises the following steps: Step 1: preparing a seed, comprising inoculating a single colony of the transformant according to claim 4 in (2) into fresh liquid medium to obtain a seed; Step 2: expanding culture, comprising inoculating the seed obtained in Step 1 into fresh liquid medium to obtain an expanded culture product; Step 3: fermentation culture, comprising adding an inducer xylose to the expanded culture product obtained in Step 2 and culturing to obtain a fermentation culture product; Step 4: washing and concentrating, comprising collecting bacterial cells and fermentation supernatant by freezing centrifugation of the fermentation culture product obtained in Step 3, and resuspending the bacterial cells to obtain the probiotic preparation according to claim 4 in (5).

7. The fish-derived antibacterial peptide according to claim 1 or 2, or the polynucleotide according to claim 3, or the agent according to claim 4, or the method according to claim 5 or 6 for use in the following applications: antibiotic substitution, intestinal health of farmed fish, fish farming, promoting fish growth, coping with pathogenic bacterial infection, improving the spread of antibiotic resistance, reducing drug residues, improving environmental pollution, reducing the risk of drug resistance, improving intestinal immunity, protecting intestinal structure, improving the intestinal inflammatory environment, restoring intestinal flora homeostasis, preparing a drug for preventing / suppressing / alleviating / reducing / treating / diagnosing a disease, developing a feed additive, developing an antibacterial peptide, heterologous expression of a fish-derived antibacterial peptide, saving farming costs.

8. The use of a non-canonical signal peptide sfGFP in the heterologous expression of a fish-derived antibacterial peptide as claimed in claim 1, characterized in that, The application comprises using the fusion protein sfGFP as a signal peptide at the N-terminus of the antibacterial peptide to guide the fish-derived antibacterial peptide to be secreted outside the cell.

9. The fish-derived antibacterial peptide, polynucleotide, composition, method, use according to any one of claims 1 to 8, wherein the fish-derived antibacterial peptide is a polypeptide of SEQ ID NO: 1 or a polypeptide having at least 80% sequence identity to SEQ ID NO:

1. The amino acid sequence of the fish-derived antibacterial peptide comprises one of the following sequences: (a) the amino acid sequence shown in SEQ ID NO: 1-9; (b) an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1-9; (c) an amino acid sequence having the same function as the amino acid sequence shown in SEQ ID NO: 1-9, with substitution, deletion, addition of one or more amino acid residues; (d) an amino acid sequence having the same function as the amino acid sequence shown in SEQ ID NO: 1-9, expressed by substitution, deletion, or addition of one or more nucleic acid bases in the coding nucleotide sequence; (e) an amino acid sequence having the same function as the amino acid sequence shown in SEQ ID NO: 1-9, encoded by a nucleotide sequence capable of hybridizing to the coding nucleotide sequence of the amino acid sequence under moderately stringent conditions; and / or, The polynucleotide sequence comprises one of the following sequences: (a) the nucleotide sequence shown in SEQ ID NO: 10-18; (b) a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity with the nucleotide sequence defined in (a); (c) a nucleotide sequence having the same function as the nucleotide sequence defined in (a), with substitution, deletion, or addition of one or more nucleic acid bases; (d) a nucleotide sequence capable of hybridizing to the nucleotide sequence defined in (a), (b), or (c) or its full-length complement under stringent conditions; or (e) a nucleotide sequence different from the nucleotide sequence defined in (a), (b), (c), or (d) due to the degeneracy of the genetic code; and / or, The nucleotide sequences of sfGFP-PF, sfGFP-PR, PF, PR, 1622-F, 1622-R, HF, HR, PH-F, PH-R, SPST-F, SPST-R, AHaerA-F, AHaerA-R, Tnf-aF, Tnf-aR, il-1b-F, il-1b-R, Cxcl8-F, Cxcl8-R, il-6-F, il-6-R, il-10-F, il-10-R, il-22-F, il-22-R, Tgfb-F, and Tgfb-R each include one of the following sequences: (a) Nucleotide sequences as shown in SEQ ID NO: 30-35 and 37-58, respectively; (b) A nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence defined in (a); (c) Nucleic acid sequences with equivalent functions formed by substitution, deletion or addition of one or more nucleic acid bases in the nucleotide sequences defined in (a); (d) A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence defined in (a), (b), or (c), or its full-length complement; or, (e) Nucleotide sequences that differ from the nucleotide sequences defined in (a), (b), (c), and (d) due to the degeneracy of the genetic codon; and / or, The sequences of the nucleotides include one of the following sequences: (a) Nucleotide sequences as shown in SEQ ID NO:22, 24; (b) A nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence defined in (a); (c) Nucleic acid sequences with equivalent functions formed by substitution, deletion or addition of one or more nucleic acid bases in the nucleotide sequences defined in (a); (d) A nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence defined in (a), (b), or (c), or its full-length complement; or, (e) Nucleotide sequences that differ from the nucleotide sequences defined in (a), (b), (c), and (d) due to the degeneracy of the genetic codon; and / or, The amino acid sequence of the polypeptide includes one of the following sequences: (a) The amino acid sequences shown in SEQ ID NO:21, 23; (b) an amino acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, 23; (c) an amino acid sequence that differs from the amino acid sequence set forth in SEQ ID NO: 21, 23 by virtue of one or more substitutions, deletions, additions, but has the same function; (d) an amino acid sequence that is expressed from a nucleotide sequence that differs from the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 21, 23 by virtue of one or more substitutions, deletions, or additions of nucleic acid bases, and has the same function; (e) a nucleotide sequence that hybridizes to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 21, 23 under moderately stringent conditions and encodes an amino acid sequence having the same function; and / or, the nucleotide sequence of the sfgfp is set forth in SEQ ID NO: 29; and / or, the nucleotide sequence of the pisidin-1 is set forth in SEQ ID NO: 25; and / or, the nucleotide sequence of the hepcidin is set forth in SEQ ID NO: 36; and / or, the nucleotide sequence of the 6xHis is set forth in SEQ ID NO: 19; and / or, the nucleotide sequence of the 6xhis is set forth in SEQ ID NO: 20; and / or, the nucleotide sequence of the aprE is set forth in SEQ ID NO: 26; and / or, the nucleotide sequence of the amyQ is set forth in SEQ ID NO: 27; and / or, the nucleotide sequence of the sacB is set forth in SEQ ID NO: 28; and / or, the sfGFP is a non-conventional signal peptide; and / or, the disease comprises pathogenic bacteria infection, intestinal inflammation, intestinal tissue damage, intestinal flora imbalance, environmental stress.

10. The fish-derived antibacterial peptide, polynucleotide, substance, method, use according to claim 9, wherein, the disease comprises elevated pathogenic bacteria load, intestinal inflammation, intestinal tissue damage, or death of the individual caused by infection with Aeromonas hydrophila.

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