A hordeum vulgare l. antimicrobial peptide sc1_966 and application thereof

Abstract

This invention discloses a method derived from Hoddle archaea ( Hodarchaeales This study describes the antimicrobial peptide SC1_966 and its applications, belonging to the field of biomedicine and aquaculture disease control technology. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO:1. This antimicrobial peptide consists of 35 amino acids, with a molecular weight of 4330.30 Daltons, containing 10 basic amino acid residues. It has a hydrophobicity of -0.543 and an isoelectric point of 10.84, making it a cationic short peptide with advantages such as good water solubility and high antimicrobial activity. Its compact structure makes it easy to synthesize and purify, facilitating large-scale production and application. Furthermore, the presence of multiple positively charged amino acid residues facilitates interaction with the negatively charged bacterial cell membrane, disrupting membrane integrity and thus exerting rapid antibacterial and bactericidal effects. This antimicrobial peptide exhibits broad-spectrum and highly efficient antimicrobial activity against common aquatic pathogens such as *Escherichia coli* and various Vibrio species. At effective antimicrobial concentrations, it shows no significant cytotoxicity to HeLa bacteria, demonstrating good biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of antimicrobial peptide technology, and in particular to a Hoddle archaea antimicrobial peptide SC1_966 and its applications. Background Technology

[0002] The increasing resistance to antimicrobial agents has created an urgent need for novel molecules to discover or design new antimicrobial agents. Antimicrobial peptides (AMPs) are mainly produced through two major biosynthetic pathways: ribosomal synthesis and non-ribosomal peptide synthesis, either directly or through post-translational processing of larger precursor proteins. AMPs possess broad-spectrum activity and low resistance induction; however, research has primarily focused on mining AMPs in the proteomes of bacteria, fungi, and eukaryotes, largely neglecting the archaea domain despite their evolutionary significance and significant biochemical diversity. Compared to bacteria and eukaryotes, archaea possess unique cellular structures, including distinct lipid membranes and metabolic pathways. These differences suggest that their encoded peptides may possess structural and mechanistic properties that allow them to differentiate from known antimicrobial agents, thus providing potential pathways to circumvent existing resistance mechanisms. Current research indicates that archaic antimicrobial peptides disrupt cell membranes by depolarizing the bacterial cytoplasmic membrane, thereby enabling rapid bacterial kill. To date, only a few archaic AMPs have been described. Halocins were among the first archaea AMPs studied. These peptides are secreted into the environment by Halococcaceae, where they kill other microorganisms in the same niche or inhibit their metabolism and growth, thereby reducing competition for basic resources such as nutrients and oxygen.

[0003] In aquaculture, bacterial diseases are a key factor restricting the industry's development, with Vibrio being particularly prominent, becoming one of the major biological threats causing significant economic losses globally. In intensive, high-density aquaculture, diseases spread rapidly, causing continuous production losses. Current prevention and control mainly rely on comprehensive management measures such as optimizing the aquaculture environment and strengthening quarantine and immunization. However, during acute outbreaks, these methods have limited effectiveness and are difficult to control quickly. In actual treatment, chemical drugs and antibiotics, such as fluoroquinolones, tetracyclines, and sulfonamides, are still widely used. Long-term excessive use has led to increasingly serious Vibrio resistance problems, with multidrug-resistant strains constantly emerging, causing many traditional drugs to lose efficacy or even become ineffective. At the same time, drug residues are a prominent problem; their accumulation in farmed organisms and the aquatic environment not only threatens food safety and consumer health but also disrupts the aquatic ecological balance, affecting the structure and function of microbial communities. With the increasing international regulation of antibiotics used in aquaculture, developing highly effective, safe, non-resistant, and environmentally friendly new antibacterial agents has become an urgent innovative direction in the field of aquatic disease control.

[0004] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a Hodarchaeales antimicrobial peptide SC1_966 and its application, aiming to solve the problems that the existing antimicrobial peptides have poor antimicrobial effects and cannot be applied to aquaculture.

[0006] The technical solution of the present invention is as follows: A Hodel archaea antimicrobial peptide SC1_966, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] An antimicrobial composition comprising the Hoddle archaea antimicrobial peptide SC1_966.

[0008] The antimicrobial composition further includes an adjuvant acceptable for aquaculture.

[0009] The antibacterial composition, wherein the aquaculture includes freshwater aquaculture.

[0010] The use of a Hoddle archaea antimicrobial peptide SC1_966 in the preparation of products for the prevention and / or treatment of bacterial infections.

[0011] In the aforementioned application, the bacteria include one or more of Escherichia coli, Vibrio parahaemolyticus, and Vibrio alginolyticus.

[0012] In the aforementioned application, the product for preventing and / or treating bacterial infections is a bacteriostatic agent or a bactericide.

[0013] In the aforementioned application, the concentration of the antibacterial agent or bactericide is above 86 μM.

[0014] Application of Hoddle archaeal antimicrobial peptide SC1_966 in the preparation of Vibrio spp. biofilm products.

[0015] A feed containing an antimicrobial peptide, said feed comprising the Hodel archaea antimicrobial peptide SC1_966.

[0016] Beneficial Effects: This invention provides a Hodel archaeal antimicrobial peptide SC1_966 and its applications. The amino acid sequence of the Hodel archaeal antimicrobial peptide SC1_966 is shown in SEQ ID NO:1. The Hodel archaeal antimicrobial peptide SC1_966 provided by this invention consists of 35 amino acids, with a molecular weight of 4330.30 Daltons, containing 10 basic amino acid residues, a hydrophobicity of -0.543, and an isoelectric point of 10.84. It is a cationic short peptide with advantages such as good water solubility and high antimicrobial activity. Its limited amino acid residue composition results in a compact structure, making it easy to synthesize and purify, facilitating large-scale production and application. Furthermore, the antimicrobial peptide contains multiple positively charged amino acid residues, which facilitates interaction with the negatively charged bacterial cell membrane, disrupting membrane integrity and thus exerting rapid antibacterial and bactericidal effects. Simultaneously, the Hodel archaeal antimicrobial peptide SC1_966 exhibits broad-spectrum antimicrobial activity against various common aquatic pathogens, effectively inhibiting bacterial growth and reproduction, and delaying or inhibiting the pathogenic process, making it suitable for bacterial disease control in aquaculture. This invention shows no significant cytotoxicity to HeLa (human cervical cancer cells) cells at effective antibacterial concentrations, demonstrating good biosafety. Attached Figure Description

[0017] Figure 1 The high-performance liquid chromatogram of the Hodel archaeal antimicrobial peptide SC1_966 prepared in Example 2; Figure 2 The mass spectrometry result is shown for the antimicrobial peptide SC1_966 of Hoddle archaea prepared in Example 2. Figure 3 The kinetic curve of bactericidal action of Hoddle archaea antimicrobial peptide SC1_966 against Escherichia coli MG1655; Figure 4 The kinetic curve of bactericidal activity of Hoddle archaea antimicrobial peptide SC1_966 against Vibrio parahaemolyticus ATCC 202005; Figure 5 The kinetic curve of bactericidal action of Hoddle archaea antimicrobial peptide SC1_966 against Vibrio alginolyticus ATCC 17749; Figure 6 The graph shows the half-maximal inhibitory concentration (IC50) of the Hoddle archaeal antimicrobial peptide SC1_966 against HeLa (human cervical cancer cells). Detailed Implementation

[0018] This invention provides a Hoddle archaea antimicrobial peptide SC1_966 and its applications. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0019] It will be understood by those skilled in the art that, unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should also be understood that terms such as those defined in general dictionaries should be understood to have the same meaning as in the context of the prior art, and should not be interpreted in an idealized or overly formal sense unless specifically defined as herein.

[0020] To date, only a few archaeal AMPs have been described. A recent study published in Nature Microbiology addresses this challenge by systematically analyzing the archaeal proteome using APEX 1.1, a state-of-the-art deep learning framework. APEX employs a multi-task learning structure to predict the antimicrobial activity of input peptide sequences. It combines an encoder neural network with multiple fully connected downstream neural networks to perform two main prediction tasks: (1) binary classification of peptides as antimicrobial or non-antimicrobial, and (2) regression-based prediction of antimicrobial activity against specific bacterial pathogens, enabling rapid screening of short peptides with potential antimicrobial capabilities. Following computational discovery, subpopulations of antimicrobial peptides were synthesized and tested in vitro on a cohort of clinically relevant samples, completing the development of an antimicrobial agent repository.

[0021] In aquaculture, disease control mainly relies on comprehensive management measures such as optimizing the aquaculture environment and strengthening quarantine and immunization. However, during acute outbreaks, these methods have limited effectiveness and are insufficient to quickly control the epidemic. Furthermore, existing antimicrobial peptides have poor antimicrobial efficacy and cannot be applied to aquaculture.

[0022] Based on this, the present invention provides a Hodel archaea antimicrobial peptide SC1_966, the amino acid sequence of which is shown in SEQ ID NO:1. Its amino acid sequence is MKKSFYNDHKIKLFKGKKILVNWRQIRISPKFLNL.

[0023] In this embodiment, the provided Hoddle archaea antimicrobial peptide SC1_966 is composed of 35 amino acids with a molecular weight of 4330.30 Daltons. It contains 10 basic amino acid residues, has a hydrophobicity of -0.543, and an isoelectric point of 10.84. As a cationic short peptide, it possesses advantages such as good water solubility and high antimicrobial activity. Its limited amino acid residue composition results in a compact structure, making it easy to synthesize and purify, facilitating large-scale production and application. Furthermore, the presence of multiple positively charged amino acid residues facilitates interaction with the negatively charged bacterial cell membrane, disrupting membrane integrity and thus exerting rapid antibacterial and bactericidal effects. Simultaneously, the Hoddle archaea antimicrobial peptide SC1_966 exhibits broad-spectrum antimicrobial activity against various common aquatic pathogens, effectively inhibiting bacterial growth and reproduction, and delaying or suppressing pathogenic processes, making it suitable for bacterial disease control in aquaculture.

[0024] Specifically, the Hodel archaeal antimicrobial peptide SC1_966 possesses broad-spectrum antimicrobial activity and a strong bactericidal rate; furthermore, its use in peptide form for microbial prevention and treatment carries extremely low risk of drug resistance and environmental pollution, demonstrating broad application prospects. The antimicrobial peptide provided by this invention exhibits broad-spectrum and highly efficient antimicrobial activity against common aquatic pathogens such as *Escherichia coli* and various Vibrio species (e.g., *Vibrio parahaemolyticus*, *Vibrio alginolyticus*), and shows no significant cytotoxicity to HeLa (human cervical cancer cells) at effective antimicrobial concentrations, demonstrating good biocompatibility. This provides a safe and residue-free novel candidate drug for the prevention and treatment of bacterial diseases in aquaculture.

[0025] In addition, the present invention also provides an antibacterial composition comprising the Hoddle archaea antimicrobial peptide SC1_966.

[0026] In this embodiment, the Hoddle archaea antimicrobial peptide SC1_966 is used as the active ingredient and is compounded with other adjuvants to form a stable antimicrobial composition. The antimicrobial peptide SC1_966 can interact with the negative charge of the bacterial cell membrane, thereby disrupting the membrane integrity and exerting rapid antibacterial and bactericidal effects. This makes the antimicrobial composition containing the antimicrobial peptide SC1_966 usable for the prevention and treatment of aquatic animal diseases and effectively improves aquaculture efficiency and product quality.

[0027] In some embodiments, the antimicrobial composition further includes an adjuvant acceptable for aquaculture.

[0028] In some embodiments, the adjuvants include, but are not limited to, sustained-release carriers, molding agents, antioxidants, enzyme preparations, prebiotic synergists, and nutritional fortifiers.

[0029] In some embodiments, the sustained-release carrier includes, but is not limited to, one or more natural minerals such as montmorillonite, bentonite, zeolite powder, and diatomaceous earth; the aforementioned sustained-release carrier can delay the release of antimicrobial peptides in water, reduce protease degradation, and improve stability in water. Preferably, the mass ratio of the sustained-release carrier to the antimicrobial peptide SC1_966 is (10-100):1.

[0030] In some embodiments, the forming agent includes, but is not limited to, one or more of the following substances with adhesive properties: sodium alginate, carrageenan, gelatin, guar gum, sodium carboxymethyl cellulose (CMC), sodium polyacrylate, etc.; it can be used to improve the cohesiveness of granules during granulation or tableting and reduce the loss of activity during feed processing.

[0031] In some embodiments, the antioxidants include, but are not limited to, one or more of vitamin C phosphate, vitamin E, butylated hydroxytoluene (BHT), tert-butylhydroquinone (TBHQ), and citric acid; the antioxidants described above can be used to block peptide chain oxidation and lipid oxidation, thereby extending shelf life.

[0032] In some embodiments, the enzyme preparation includes, but is not limited to, thermostable phytase, protease, lipase; one or more of citric acid, fumaric acid, calcium formate, and sodium butyrate; which can lower the gastrointestinal pH, inhibit pathogens, and at the same time reduce the destruction of antimicrobial peptides by endogenous proteases, thereby improving the absorption rate.

[0033] In some embodiments, the prebiotic synergist includes, but is not limited to, Bacillus subtilis, Bacillus brevis, lactic acid bacteria, yeast culture; one or more of inulin, fructooligosaccharides, and mannooligosaccharides; which can form a "bactericidal-occupation-immunity" triple synergistic effect with antimicrobial peptides, reducing the risk of drug resistance.

[0034] In some embodiments, the nutritional fortifier includes, but is not limited to, one or more of β-glucan, chitosan oligosaccharide, taurine, arginine, N-carbamoylglutamic acid (NCG), and krill oil (rich in EPA / DHA); it can activate the non-specific immunity of fish and shrimp, promote wound healing, and form a dual pathway of "antibacterial + immune enhancement" with antimicrobial peptides.

[0035] In some embodiments, the aquaculture includes freshwater aquaculture. Applying the Hoddle archaea antimicrobial peptide SC1_966 to aquaculture pest control exhibits advantages such as good water solubility and high antibacterial activity. Furthermore, the positively charged amino acid residues facilitate interaction with the negatively charged bacterial cell membrane, disrupting membrane integrity and thus exerting rapid antibacterial and bactericidal effects.

[0036] In a preferred embodiment, the freshwater aquaculture species includes Litopenaeus vannamei; the Hoddle archaea antimicrobial peptide SC1_966 is used as an antimicrobial agent for aquaculture, particularly for Litopenaeus vannamei (…). Litopenaeus vannamei ) Scenarios for the prevention and control of bacterial diseases during the seedling and adult shrimp stages.

[0037] In addition, the present invention provides the use of Hoddle archaeal antimicrobial peptide SC1_966 in the preparation of products for the prevention and / or treatment of bacterial infections.

[0038] In this embodiment, the provided Hoddle archaea antimicrobial peptide SC1_966 contains multiple positively charged amino acid residues, which facilitate interaction with the negatively charged bacterial cell membrane, disrupting membrane integrity and thus exerting rapid antibacterial and bactericidal effects. Simultaneously, the Hoddle archaea antimicrobial peptide SC1_966 exhibits broad-spectrum antimicrobial activity against a variety of common aquatic pathogens, effectively inhibiting bacterial growth and reproduction, and delaying or suppressing the pathogenic process, making it suitable for bacterial disease control in aquaculture.

[0039] Specifically, the Hodel archaeal antimicrobial peptide SC1_966 exhibits a low minimum inhibitory concentration (MIC) against Vibrio spp., MIC ≤ 86 μM; it can be used as an antimicrobial agent in aquaculture, especially for Litopenaeus vannamei (Sinapis albus). Litopenaeus vannamei ) Scenarios for the prevention and control of bacterial diseases during the seedling and adult shrimp stages.

[0040] In some embodiments, the bacteria include one or more of *Escherichia coli*, *Vibrio parahaemolyticus*, and *Vibrio alginolyticus*. The *Hodler archaeal* antimicrobial peptide SC1_966 exhibits broad-spectrum antimicrobial activity against a variety of common aquatic pathogens (including *Escherichia coli* and *Vibrio*), effectively inhibiting bacterial growth and reproduction, and delaying or suppressing the pathogenic process, making it suitable for bacterial disease control in aquaculture.

[0041] In some embodiments, the Hoddle archaeal antimicrobial peptide SC1_966 is used in the preparation of products for the prevention and / or treatment of Vibrio parahaemolyticus infections.

[0042] In some embodiments, the product for preventing and / or treating bacterial infections is a bacteriostatic agent or a bactericide.

[0043] In some embodiments, the concentration of the antibacterial agent or bactericide is above 86 μM. Antibacterial agents or bactericides at concentrations above this level have good antibacterial and bactericidal effects, effectively inhibiting bacterial growth and reproduction, delaying or inhibiting the pathogenic process, and are suitable for bacterial disease control in aquaculture.

[0044] In addition, the present invention also provides a feed containing an antimicrobial peptide, the feed comprising the Hodel archaea antimicrobial peptide SC1_966.

[0045] In this embodiment, the Hoddle archaeal antimicrobial peptide SC1_966 is applied in aquatic feed as an additive, which can improve the antimicrobial properties of the feed, improve animal immunity and growth performance, and promote aquaculture.

[0046] Finally, the present invention also provides the application of Hodel archaeal antimicrobial peptide SC1_966 in the preparation of drugs for the prevention and / or treatment of human cervical cancer cells; it has no significant cytotoxicity to HeLa (human cervical cancer cells) at effective antimicrobial concentrations and has good biosafety.

[0047] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0048] Example 1 This embodiment uses a deep learning algorithm to screen the Hoddle archaea antimicrobial peptide SC1_966. The specific steps are as follows: Using the existing APEX 1.1 deep learning model, the minimum inhibitory concentration (MIC) against eight pathogens can be predicted based on the primary and secondary structures of proteins. By collecting proteomics data of all extant archaea with lengths ≤50 amino acids, and after model prediction, candidate peptides were ranked by activity, prioritizing highly active peptides with a predicted average MIC ≤200 μM, ultimately identifying 10,050 candidate peptides. Further screening, based on the lowest MIC and eukaryotic origin, yielded one archaeological antimicrobial peptide named SC1_966, with the amino acid sequence MKKSFYNDHKIKLFKGKKILVNWRQIRISPKFLNL (SEQ ID NO:1). The MIC prediction data of antimicrobial peptide SC1_966 against pathogenic bacteria are shown in Table 1.

[0049] Table 1. MIC prediction data of antimicrobial peptide SC1_966 against pathogenic bacteria

[0050] Example 2 Archaea antimicrobial peptide SC1_966 with a purity of over 90% can be obtained using existing solid-phase chemical synthesis methods. In this embodiment, the Hoddle archaea antimicrobial peptide SC1_966 was synthesized by Suzhou Hongxun Biotechnology Co., Ltd. using a solid-phase synthesis method. High-performance liquid chromatography (HPLC) and mass spectrometry detection information are as follows: Figure 1 and Figure 2 As shown in Table 2, the physicochemical parameters of the Hoddle archaea antimicrobial peptide SC1_966 are as follows: Table 2 Physicochemical parameters of Hoddle archaea antimicrobial peptide SC1_966

[0051] Example 3 The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCC) of the Hoddle archaea antimicrobial peptide SC1_966 were determined, including: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the Hoddle archaeal antimicrobial peptide SC1_966 obtained in Example 2 were determined. The strains involved were: *Escherichia coli* MG1655, *Vibrio parahaemolyticus* ATCC 202005, and *Vibrio alginolyticus* ATCC 17749. *Escherichia coli* MG1655 was purchased from the Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences, while *Vibrio parahaemolyticus* ATCC 202005 and *Vibrio alginolyticus* ATCC 17749 were both purchased from the China Marine Microbial Culture Collection Center.

[0052] The specific method is as follows: The preserved *E. coli* were plated on nutrient broth plates and incubated upside down at 37°C overnight, representing the F1 generation. *Vibrio parahaemolyticus* and *Vibrio alginolyticus* were plated on 2216E plates and incubated upside down at 28°C overnight, representing the F1 generation. The next day, single colonies of each were picked and streaked again on nutrient broth plates or 2216E plates for subculturing overnight, representing the F2 generation.

[0053] Colonies were picked from each F2 plate and inoculated into the corresponding liquid culture medium. The plates were then placed in 96-well plates, and OD changes were monitored in real time using a growth curve analyzer. When the OD reached 1.5-2.0, the plates were diluted 100-fold with the corresponding liquid culture medium to achieve a final bacterial concentration of 5 × 10⁻⁶. 5 Approximately CFU / mL.

[0054] Dissolve and dilute the SC1_966 powder with sterile MilliQ water to the storage concentration, to 516 or 688 μM, and store on ice for later use.

[0055] Add 5×10⁻⁶ cells to wells 1-3 of row A in a 96-well cell culture plate. 5CFU / mL bacterial suspension; add the corresponding liquid culture medium to wells 4-6; add 2-fold concentration of the corresponding liquid culture medium to wells 7-10; add the test protein sample (SC1_966) and the corresponding liquid culture medium to wells 11-12. Add 100 μL of the corresponding drug solution to well 1 in row BH; add 50 μL of 2-fold concentration of the corresponding liquid culture medium to wells 2-12 in row BH. First, use a multi-channel pipette to serially dilute the corresponding drug solution 11 times from wells BH and wells 1-11, to concentrations of 258 μM, 129 μM, 64.5 μM, 32.25 μM down to 0.25 μM; or to concentrations of 344 μM, 172 μM, 86 μM, 43 μM down to 0.34 μM; then add 50 μL of the test bacterial suspension to wells BH and wells 1-11; add 50 μL of sterile MilliQ water to wells BH and well 12. For each type of bacteria to be tested, a blank control group, a negative control group, a positive control group, and an experimental group were set up, with three replicates for each group: a. Blank control group: 50 μL of the protein sample to be tested (SC1_966) and 50 μL of culture medium; negative control group: 100 μL bacterial suspension; c. Positive control group: 50 μL polymyxin B and 50 μL bacterial suspension; Experimental group d: 50 μL of the protein sample to be tested (SC1_966) and 50 μL of bacterial suspension.

[0056] The 96-well cell culture plate was placed in a bacterial growth curve instrument at a suitable temperature, and the OD changes were monitored in real time and cultured for 24 hours. The MIC results of the experimental group were observed. The experimental group was gently mixed by pipetting, and an appropriate amount of bacterial solution was taken for serial dilution and spread on the corresponding solid culture medium plates. The plates were incubated upside down at a suitable temperature for 24 hours, and the MBC results were observed.

[0057] The antibacterial activity results of SC1_966 obtained in this embodiment are shown in Table 3, indicating that it has broad-spectrum antibacterial activity.

[0058] Table 3. Antimicrobial activity of antimicrobial peptide SC1_966

[0059] Note: MIC: Minimum inhibitory concentration (μM), denoted by a. a: The highest protein concentration at which bacterial growth is visible to the naked eye; MBC: Minimum bactericidal concentration (μM), denoted by b, b: The lowest protein concentration at which no bacterial growth is visible to the naked eye. b represents the lowest protein concentration capable of killing 99.9% of bacteria.

[0060] Example 4 This embodiment investigates the bactericidal kinetics of the Hoddle archaea antimicrobial peptide SC1_966, as detailed below: In this embodiment, Escherichia coli MG1655, Vibrio parahaemolyticus ATCC 202005, and Vibrio alginolyticus ATCC 17749 were selected as test bacteria to determine the bactericidal kinetics of the Hoddle archaeal antimicrobial peptide SC1_966 obtained in Example 2.

[0061] The specific method is similar to the antibacterial activity assay described in Example 3. The final concentration of SC1_966 was adjusted to 1 times and 1.3 times the minimum bactericidal concentration (MBC) using the corresponding liquid culture medium, and an appropriate time point for co-incubation with the antimicrobial peptide was set. Then, 10 μL of the incubation solution was pipetted onto the corresponding solid plates, and the number of colonies was counted. The bactericidal kinetic curve of the antimicrobial peptide SC1_966 against Escherichia coli MG1655 is shown below. Figure 3 As shown, the bactericidal kinetics curve of antimicrobial peptide SC1_966 against Vibrio parahaemolyticus ATCC202005 is as follows. Figure 4 As shown, the bactericidal kinetic curve of antimicrobial peptide SC1_966 against Vibrio alginolyticus ATCC 17749 is as follows. Figure 5 As shown.

[0062] It can be seen that SC1_966 can kill 99.99% of Escherichia coli and Vibrio alginolyticus at a final concentration of 258 μM in 0.5 h; SC1_966 can kill Vibrio parahaemolyticus reduced by 3 orders of magnitude in 1 h at a final concentration of 258 μM, and can kill 99.99% of Vibrio parahaemolyticus in 2 h.

[0063] Example 5 In this embodiment, the CCK-8 assay was used to determine the half-maximal inhibitory concentration (IC50) of the antimicrobial peptide SC1_966 against HeLa (human cervical cancer cells) cells. The cytotoxicity of the antimicrobial peptide SC1_966 obtained in Example 1 was also determined. The specific steps are as follows: (1) Cell resuscitation and plating: Observe HeLa cell culture dishes under an inverted microscope. When 80%-90% of the cells in the field of view are observed to be adhered and growing in an island-like pattern, perform passage treatment. Discard the old culture medium in the culture dish, add HBSS buffer to rinse 1-2 times and discard the old medium, add an appropriate amount of trypsin digestion solution to digest for 1-2 min. When the cell cytoplasm shrinks and the intercellular space increases, add DMEM complete culture medium containing serum to stop digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Resuspend the cells in fresh DMEM complete culture medium, count them using a cell counting chamber, and adjust the cell concentration to 1×10⁶ cells / mL. 5 Cells / mL. Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate in a cell culture incubator at 37°C and 5% CO2 saturated humidity until the cell adhesion rate reaches more than 80%.

[0064] (2) Drug addition treatment: Carefully aspirate the old culture medium from the wells. Add 100 μL of culture medium containing different gradient concentrations (64.5 μM, 86.0 μM, 129.0 μM, 172.0 μM, 258.0 μM, 344.0 μM) of antimicrobial peptide SC1_966 to the experimental groups; set up 3 parallel replicates for each concentration. At the same time, set up complete culture medium without antimicrobial peptide as a negative control group and cell-free culture medium as a blank group. Place the culture plate in a cell culture incubator and continue to culture for 24 h.

[0065] (3) Absorbance measurement: After the culture is completed, add 10 μL of CCK-8 solution to each well, gently shake to mix, and incubate in a cell culture incubator at 37℃ in the dark for 1 h. Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0066] (4) Data processing and result analysis: The formula for calculating cell viability is: Cell viability (%) = [(OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group)] × 100%. Experimental results are as follows: Figure 6 As shown, the cytotoxicity of the antimicrobial peptide SC1_966 exhibits a significant threshold effect. At concentrations of 86 μM and below, the survival rate of HeLa cells remains above 100%, demonstrating good biocompatibility and no significant cytotoxicity. When the concentration increases to 129 μM (corresponding to 1.5 MIC) and above, the cell survival rate significantly decreases to approximately 35%. Nonlinear regression fitting calculations indicate that the IC50 of the antimicrobial peptide SC1_966 against HeLa cells is approximately 91.31 μM. Based on these results, although high concentrations exhibit some cytotoxicity, in practical Vibrio control applications, it is sufficient to keep the Vibrio biomass in the environment below the pathogenicity threshold to meet production needs; it is not necessarily necessary to pursue extremely high concentrations for complete eradication. Therefore, in practical applications, it is recommended to preferentially use a dose of 86 μM or lower. This dose can effectively inhibit Vibrio growth while ensuring no toxicity to host cells, reflecting the flexibility and safety of the dosage selection in this invention.

[0067] In summary, this invention provides a Hodel archaeal antimicrobial peptide SC1_966 and its applications. The amino acid sequence of the Hodel archaeal antimicrobial peptide SC1_966 is shown in SEQ ID NO:1. The Hodel archaeal antimicrobial peptide SC1_966 provided by this invention consists of 35 amino acids, has a molecular weight of 4330.30 Daltons, contains 10 basic amino acid residues, has a hydrophobicity of -0.543, and an isoelectric point of 10.84. It is a cationic short peptide with advantages such as good water solubility and high antimicrobial activity. Its limited amino acid residue composition results in a compact structure, making it easy to synthesize and purify, facilitating large-scale production and application. Furthermore, this antimicrobial peptide contains multiple positively charged amino acid residues, which facilitates interaction with the negatively charged bacterial cell membrane, disrupting membrane integrity and thus exerting rapid antibacterial and bactericidal effects. Meanwhile, the Hoddle archaeal antimicrobial peptide SC1_966 exhibits broad-spectrum antimicrobial activity against a variety of common aquatic pathogens, effectively inhibiting bacterial growth and reproduction, and delaying or inhibiting the pathogenic process, making it suitable for bacterial disease control scenarios in aquaculture.

[0068] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A Hoddle archaea antimicrobial peptide SC1_966, characterized in that, The amino acid sequence of the Hodel archaeal antimicrobial peptide SC1_966 is shown in SEQ ID NO:

1.

2. An antibacterial composition, characterized in that, The antimicrobial composition comprises the Hoddle archaea antimicrobial peptide SC1_966 as described in claim 1.

3. The antibacterial composition according to claim 2, characterized in that, The antimicrobial composition also includes adjuvants acceptable for aquaculture.

4. The antibacterial composition according to claim 3, characterized in that, The aquaculture mentioned includes freshwater aquaculture.

5. The use of the Hoddle archaeal antimicrobial peptide SC1_966 as described in claim 1 in the preparation of products for the prevention and / or treatment of bacterial infections.

6. The application according to claim 5, characterized in that, The bacteria include one or more of Escherichia coli, Vibrio parahaemolyticus, and Vibrio alginolyticus.

7. The application according to claim 5, characterized in that, The products for the prevention and / or treatment of bacterial infections are bacteriostatic agents or bactericides.

8. The application according to claim 7, characterized in that, The concentration of the antibacterial agent or bactericide is above 86 μM.

9. A feed containing antimicrobial peptides, characterized in that, The feed comprises the Hoddle archaea antimicrobial peptide SC1_966 as described in claim 1.

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