Antimicrobial peptide, synthesis method and application thereof

Abstract

The present invention discloses an antimicrobial peptide, its synthesis method and application, and belongs to the field of antimicrobial peptides. The sequence of the antimicrobial peptide is: F 23 IHHIFNGIANVGRMIHGIV 42 The present invention relates to a method for synthesizing an antimicrobial peptide, comprising the steps of obtaining a full-length cDNA of the Piscidin gene, designing the antimicrobial peptide, and synthesizing the antimicrobial peptide. The present invention is applicable to the antimicrobial treatment of largemouth bass, addressing the problem of existing largemouth bass disease prevention using antibiotics, which leads to drug resistance and water pollution, thus hindering the development of the largemouth bass aquaculture industry. The invention exhibits strong specific immunity against Aeromonas hydrophila and Vibrio splendens.

Description

Technical Field

[0001] The present invention belongs to the field of antimicrobial peptides, and in particular relates to an antimicrobial peptide, a synthesis method and application thereof. Background Art

[0002] Antimicrobial peptides (AMPs), also known as antimicrobial peptides and host defense peptides (HDPs), are a class of small, biologically active peptides widely found in nature. They are also a key component of the body's innate immunity. AMPs possess broad-spectrum antimicrobial activity, with strong inhibitory effects against Gram-negative and Gram-positive bacteria, as well as fungi. Some AMPs also possess anti-tumor activity and can regulate inflammatory responses. They are known as "natural antibiotics" for their non-polluting, residue-free, broad-spectrum antimicrobial properties, and resistance to drug resistance. They are expected to replace antibiotics in the prevention and control of pathogenic diseases in aquatic animals.

[0003] Antimicrobial peptides are primarily obtained through three methods: extraction of endogenous antimicrobial peptides from organisms, chemical synthesis, and genetically engineered expression. To date, the antimicrobial peptides discovered in fish primarily include piscidins, β-defensins, hepcidins, cathelicidins, liver-expressed antimicrobial peptides 2 (LEAP-2), and NK-lysins. Piscidins are among the most common antimicrobial peptides in fish and are further subdivided into pleurocidins, misgurins, piscidins, moronecidins, epinecidins, dicentracins, and gaduscidins. These peptide subtypes typically consist of 18-27 amino acid residues and exhibit an α-helical structure. They belong to the same evolutionarily related family, but exhibit relatively low sequence homology. Based on sequence similarity and phylogenetic analysis, piscidin subtypes are further classified into three categories: piscidin-1, piscidin-2, and piscidin-3. The main difference between them is that the Piscidin-1 sequence is longer than Piscidin-2 and Piscidin-3 and encodes an additional region rich in hydrophilic amino acids (glutamic acid and aspartic acid) at its C-terminus. The mature peptide sequences of Piscidin-2 and Piscidin-3 are similar in length and amino acid composition, but the Piscidin-2 mature peptide has an HR / K proprotein convertase cleavage motif at the C-terminus, while the Piscidin-3 cleavage motif is RRRH.

[0004] Largemouth bass is a major freshwater aquaculture species in my country. With the expansion of aquaculture and increasing intensification, bacterial and viral diseases have become increasingly common during their growth. Currently, antibiotics are the primary preventative treatment. However, long-term use can lead to varying degrees of drug resistance, resulting in economic losses and environmental pollution, severely hindering the development of the largemouth bass aquaculture industry. Research on the immune response of largemouth bass to bacterial and viral infection is limited, and the molecular mechanisms and mechanisms of action of piscidins remain largely unknown, significantly limiting the development of immunological approaches to disease prevention. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is to overcome the problem that the existing largemouth black bass use antibiotics to prevent diseases, which causes drug resistance and water environmental pollution, thereby restricting the development of the largemouth black bass breeding industry. An antimicrobial peptide with strong specific immune function against Aeromonas hydrophila and Vibrio splendens, its synthesis method and application are proposed.

[0006] In order to solve the technical problem, the technical solution adopted by the present invention is:

[0007] In one aspect, the present invention provides an antimicrobial peptide, the sequence of which is: F 23 IHHIFNGIANVGRMIHGIV 42 .

[0008] Another aspect of the present invention provides a method for synthesizing the antimicrobial peptide, comprising the steps of obtaining the full-length cDNA of the Piscidin gene, designing the antimicrobial peptide, and synthesizing the antimicrobial peptide.

[0009] Preferably, the antimicrobial peptide design step comprises: predicting the N-terminal signal peptide based on the amino acid sequence of MsPiscidin-3 using SignalP5.0, predicting the protein secondary structure using PSIPRED 4.0, predicting the protein physicochemical properties using the ProtParam tool, analyzing the molecular weight, isoelectric point, fat coefficient, instability coefficient and hydrophilicity coefficient, and designing and modifying MsPiscidin-3 based on protein secondary structure simulation, focusing on the key elements of signal peptide, antimicrobial 12 domain and protease cleavage site, and determining the F 23 IHHIFNGIANVGRMIHGIV 42 The sequence is used as the MsPiscidin-3 prolonging peptide.

[0010] Preferably, the step of obtaining the full-length cDNA of the Piscidin gene comprises: cloning and obtaining the full-length cDNA of the largemouth bass antimicrobial peptide Piscidin gene using RT-PCR and RACE-PCR methods.

[0011] Preferably, the step of obtaining the full-length cDNA of the Piscidin gene specifically comprises: taking spleen tissue of largemouth bass, extracting total RNA by the Trizol method, using the total RNA as a template, and synthesizing the first-strand cDNA, 5'RACE and 3'RACE cDNA of largemouth bass using a reverse transcription kit and a RACE cDNA amplification kit respectively;

[0012] Using the first strand of cDNA as a template, the middle fragment sequence of the Piscidin gene of largemouth bass was obtained through PCR amplification, product recovery and sequencing.

[0013] According to the results of intermediate fragment sequencing, 5'RACE and 3'RACE primers were designed, and 5'RAC cDNAE and 3'RACE cDNA were used as templates, respectively. PCR amplification, product recovery, and sequencing were also performed to obtain 5'RACE and 3'RACE gene fragments.

[0014] The sequence results obtained by sequencing were spliced ​​using Seqman software, and the corresponding sequences of other species in the database were searched, aligned, and analyzed using the BLAST service of NCBI;

[0015] The corresponding amino acid sequences were deduced using the translation tool on the ExPASy website. The amino acid sequence homology between different species was analyzed using MatGAT 2.02 software. Protein domains were predicted using the SMART online tool and the conserved domain prediction service of NCBI. A phylogenetic tree was constructed using MEGA 11 software based on the neighbor-joining method with a bootstrapping value of 10,000.

[0016] Preferably, the step of synthesizing the antimicrobial peptide comprises: synthesizing a MsPiscidin-3 derivative peptide as the antimicrobial peptide using Fmoc solid phase synthesis method.

[0017] Preferably, the steps of synthesizing the antimicrobial peptide include, in sequence: activation of the resin, condensation of amino acids, cleavage of the polypeptide, and purification and identification of the polypeptide.

[0018] The present invention also provides the application of the antimicrobial peptide in antibacterial treatment of largemouth bass.

[0019] Preferably, the antimicrobial peptide is used as an antimicrobial drug for the antibacterial effect of largemouth bass.

[0020] Preferably, the antimicrobial peptide is used as an antimicrobial drug for preventing and treating hemorrhagic disease and septicemia of largemouth bass caused by Aeromonas hydrophila infection, and ulcerous bacterial disease of largemouth bass caused by Vibrio splendens.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides an antimicrobial peptide, which has a strong inhibitory effect on Aeromonas hydrophila and also has a certain inhibitory effect on Vibrio splendens, indicating that the MsPiscidin-3 derivative peptide has strong specific antimicrobial activity and is expected to be applied to antimicrobial drugs to replace antibiotics for preventing and treating bacterial diseases such as hemorrhagic disease and septicemia caused by Aeromonas hydrophila infection and ulcer disease caused by Vibrio splendens, thereby avoiding symptoms such as skin rot and ulcers, visceral enlargement and congestion, and bleeding in fish, thereby providing a safe and environmentally friendly source of disease prevention and control for largemouth sea bass farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Figure 3 is the full-length cDNA sequence of the Piscidin-3 gene of largemouth bass and its corresponding amino acid sequence, in which the start codon (ATG) and stop codon (TAG) are marked with boxes, and the stop codon (TGA) in the box is highlighted in gray; the polyadenylation signal (AATAAA) is bolded; the N-terminal signal peptide is highlighted in yellow, the active peptide is highlighted in green, the antibacterial 12 domain is bolded and underlined in red, and the proprotein convertase cleavage motif is boxed;

[0024] Figure 2 This is a phylogenetic analysis of Piscidins in different teleost fishes. Blue, red, and green represent Piscidins-1, Piscidins-2, and Piscidins-3, respectively. The gene accession number is listed after the species name.

[0025] Figure 3 HPLC analysis of Piscidin-3-derived peptides from largemouth bass;

[0026] Figure 4 Mass spectrometry analysis of peptides derived from Piscidin-3 in largemouth bass. DETAILED DESCRIPTION

[0027] The following is a detailed and complete description of the technical solutions in the specific embodiments of the present invention. Obviously, the described embodiments are only some specific implementation methods of the overall technical solution of the present invention, and are not all implementation methods. Based on the overall concept of the present invention, all other embodiments obtained by ordinary skill in the art are within the scope of protection of the present invention.

[0028] On the one hand, the present invention provides an antimicrobial peptide, the sequence of which is: F 23 IHHIFNGIANVGRMIHGIV 42The antimicrobial peptide has a fat coefficient of 131.50 and an instability index of 14.13, making it much more stable than the original peptide. It is easy to synthesize and use in subsequent activity analysis and applications. The antimicrobial peptide has a strong inhibitory effect against Aeromonas hydrophila and also has a certain inhibitory effect against Vibrio splendens, indicating that the MsPiscidin-3 derivative peptide has strong specific antimicrobial activity and is expected to be used in antimicrobial drugs.

[0029] Another aspect of the present invention provides a method for synthesizing the antimicrobial peptide, comprising the steps of obtaining the full-length cDNA of the Piscidin gene, designing the antimicrobial peptide, and synthesizing the antimicrobial peptide.

[0030] The antimicrobial peptide design steps include: predicting the N-terminal signal peptide based on the amino acid sequence of MsPiscidin-3 using SignalP5.0, predicting the protein secondary structure using PSIPRED 4.0, predicting the protein physicochemical properties using the ProtParam tool, analyzing the molecular weight, isoelectric point, fat coefficient, instability coefficient and hydrophilicity coefficient, and designing and modifying MsPiscidin-3 based on the protein secondary structure simulation, focusing on the key elements such as signal peptide, antimicrobial 12 domain and protease cleavage site, and determining the F 23 IHHIFNGIANVGRMIHGIV 42 The sequence is called MsPiscidin-3 prolonging peptide.

[0031] Furthermore, the step of obtaining the full-length cDNA of the Piscidin gene includes: cloning and obtaining the full-length cDNA of the largemouth bass antimicrobial peptide Piscidin gene using RT-PCR and RACE-PCR methods. Specifically: spleen tissue of largemouth bass was obtained, and total RNA was extracted using the Trizol method. Using this as a template, a reverse transcription kit and a RACE cDNA amplification kit were used to synthesize the first-chain cDNA, 5'RACE and 3'RACE cDNA of largemouth bass respectively; using the first-chain cDNA as a template, the intermediate fragment sequence of the Piscidin gene of largemouth bass was obtained through PCR amplification, product recovery and sequencing; 5'RACE and 3'RACE primers were designed based on the sequencing results of the intermediate fragment, and 5'RAC cDNAE and 3'RACE cDNA were used as templates respectively, and 5'RACE and 3'RACE gene fragments were obtained through PCR amplification, product recovery and sequencing methods; the sequence results obtained by sequencing were spliced ​​using Seqman software, and the corresponding sequences of other species in the database were searched, compared and analyzed using the BLAST service of NCBI; the corresponding amino acid sequence was deduced using the translation tool on the ExPASy website; MatGAT 2.02 software was used to analyze the amino acid sequence homology among different species; the protein domains were predicted using the SMART online tool and the conserved domain prediction service of NCBI; and the phylogenetic tree was constructed using the neighbor-joining method using MEGA11 software with a bootsraps value of 10,000.

[0032] In a preferred embodiment, the step of synthesizing the antimicrobial peptide includes: synthesizing a MsPiscidin-3 derivative peptide as the antimicrobial peptide using Fmoc solid-phase synthesis, which includes: resin activation, amino acid condensation, peptide cleavage, and peptide purification and identification.

[0033] The above-mentioned antimicrobial peptide synthesis method uses gene cloning technology to obtain a largemouth bass antimicrobial peptide cDNA full length, which is named MsPiscidin-3 according to sequence analysis. Then, based on protein structure simulation, combined with factors such as signal peptide and antimicrobial 12 domain, MsPiscidin-3 is designed and modified to intercept F 23 IHHIFNGIANVGRMIHGIV 42 MsPiscidin-3 prolonging peptide was further synthesized, and the antibacterial activity of the synthetic peptide was verified, showing that it has strong specific immune function against Aeromonas hydrophila and Vibrio splendens.

[0034] The present invention also provides the use of the aforementioned antimicrobial peptides for antibacterial purposes in largemouth bass. Specifically, the antimicrobial peptides are used as antimicrobial agents for antibacterial purposes in largemouth bass. Furthermore, the antimicrobial peptides are used as antimicrobial agents for preventing and treating bacterial diseases in largemouth bass such as hemorrhagic disease and septicemia caused by Aeromonas hydrophila, and ulcerative ulcers caused by Vibrio splendens. It should be noted that the above-mentioned MsPiscidin-3 derivative peptide has a strong inhibitory effect on Aeromonas hydrophila and also has a certain inhibitory effect on Vibrio splendens; the inhibitory effect on Staphylococcus aureus, Edwardsiella tarda, etc. is weak, indicating that the MsPiscidin-3 derivative peptide has strong specific antibacterial activity and is expected to be used in antibacterial drugs to replace antibiotics for the prevention and treatment of bacterial diseases such as hemorrhagic disease and septicemia caused by Aeromonas hydrophila infection, and ulcers caused by Vibrio splendens, to avoid symptoms such as skin rot and ulcers, visceral enlargement and congestion, and bleeding in fish, providing a safe and environmentally friendly source of disease prevention and control for largemouth black bass farming, with the advantages of safety, high efficiency, pollution-free and environmental protection.

[0035] In order to more clearly and in detail introduce the antimicrobial peptides provided by the embodiments of the present invention, their synthesis methods and applications, they will be described below in conjunction with specific examples.

[0036] Example

[0037] Experimental animal preparation: Healthy largemouth bass (weight 50.0±5.0g) were temporarily raised in a laboratory recirculating aquaculture system. The water temperature was maintained at 25.0±3.0℃, the photoperiod was 14h light / 10h dark, and they were fed twice a day at a fixed time and place.

[0038] 1. Cloning and sequence analysis of the Piscidin gene in largemouth bass

[0039] (1) Gene cloning steps

[0040] The full-length cDNA of the Piscidin gene, an antimicrobial peptide from largemouth bass, was cloned using RT-PCR and RACE-PCR methods. Specifically, spleen tissue from largemouth bass was obtained, and total RNA was extracted using the Trizol method. This RNA was used as a template to synthesize the first-strand, 5'RACE, and 3'RACE cDNAs of largemouth bass using a reverse transcription kit and a RACE cDNA amplification kit, respectively. Using the first-strand cDNA as a template, PCR amplification, product recovery, and sequencing were performed to obtain the intermediate fragment sequence of the Piscidin gene of largemouth bass. Based on the sequencing results of the intermediate fragment, 5'RACE and 3'RACE primers were designed. Using 5'RAC cDNA E and 3'RACE cDNA as templates, PCR amplification, product recovery, and sequencing were also performed to obtain the 5'RACE and 3'RACE gene fragments.

[0041] (2) Sequence analysis process

[0042] Sequences obtained were assembled using Seqman software, and the corresponding sequences from other species in the NCBI BLAST service were searched, aligned, and analyzed. The corresponding amino acid sequences were deduced using the translation tool on the ExPASy website. Amino acid sequence homology between species was analyzed using MatGAT 2.02 software. Protein domains were predicted using the SMART online tool and the NCBI conserved domain prediction service. A phylogenetic tree was constructed using MEGA11 software using the neighbor-joining (NJ) method with a bootstrapping value of 10,000.

[0043] (3) Results

[0044] like Figure 1 As shown in Figure 1, the largemouth bass Piscidin cDNA sequence is 401 bp long and encodes 63 amino acids. Figure 2 As shown, based on phylogenetic tree analysis, the cloned antimicrobial peptide gene was classified as Piscidin-3 and named MsPiscidin-3. Domain prediction revealed that MsPiscidin-3 possesses a 12-antimicrobial domain. Studies have shown that this 12-antimicrobial domain primarily utilizes its amphipathic α-helical structure to penetrate bacterial cell membranes, playing a crucial role in antimicrobial activity.

[0045] 2. MsPiscidin-3 structure prediction and peptide design

[0046] Based on the translated amino acid sequence of MsPiscidin-3, SignalP 5.0 was used to predict the N-terminal signal peptide, PSIPRED 4.0 was used to predict the protein secondary structure, and the ProtParam tool was used to predict the protein's physicochemical properties. The molecular weight, isoelectric point, fat coefficient, instability coefficient, and hydrophilicity were analyzed to determine the synthesis target. Based on the amino acid sequence and structural characteristics of MsPiscidin-3, MsPiscidin-3 derivative peptides were designed. According to the protein secondary structure simulation, the key elements of signal peptide, antibacterial 12 domain and protease cleavage site were considered, and the design and modification of MsPiscidin-3 were selected. 23 IHHIFNGIANVGRMIHGIV 42 The sequence was used as the MsPiscidin-3 prolonging peptide. As shown in Table 1, the prolonging peptide has a fat coefficient of 131.50 and an instability index of 14.13, which is much more stable than the original peptide and is easy to synthesize and use in subsequent activity analysis and applications.

[0047] Table 1 Comparison of physicochemical characteristics of largemouth bass Piscidin-3 and its derived peptides

[0048]

[0049] 3. Solid-phase synthesis of Piscidin-3 prolonging peptide from largemouth bass

[0050] The MsPiscidin-3 derivative peptide was synthesized using the Fmoc solid-phase synthesis method on a Tetras peptide synthesizer and separated and purified by preparative HPLC. Specific steps:

[0051] (1) Activation of resin

[0052] Resin swelling: 1 g of Fmoc-Rink-Amide-AM resin was placed in a solid phase reactor, washed with DCM, filtered, and then added with 10 ml of DCM and soaked for 30 min to allow the resin to fully swell, then filtered to remove excess solvent;

[0053] Removal of Fmoc protecting group: Add 10 ml of 20% piperidine / DMF to wash, filter, add 10 ml of 20% piperidine / DMF to wash, shake to mix, and stir for 30 minutes; drain, add DMF, isopropanol, and DMF in sequence, shake thoroughly and rinse several times, and drain.

[0054] Kaiser test: Dip a small amount of resin into a small tube and add ninhydrin / ethanol, phenol / ethanol, and KCN / pyridine dropwise in sequence. Heat in a boiling water bath. If the resin particles turn blue-purple, deprotection is successful.

[0055] (2) Condensation of amino acids

[0056] Dissolve Fmoc-AA-OH and HOBt in DMF, shake and mix thoroughly, then add DCC and shake and mix thoroughly;

[0057] The above solution was added to the solid phase reactor and reacted at room temperature for 3 h;

[0058] Rinse with DMF, isopropanol, and DMF several times, and drain;

[0059] Perform the ninhydrin test according to the method in (1): if the resin particles do not show color, it indicates that the condensation is complete; if the resin shows color, repeat the process.

[0060] The Fmoc protecting group was removed according to the method in (1), and amino acids were added sequentially with reference to the sequence of the MsPiscidin-3 derived peptide, and the above process was repeated until all amino acids were successfully condensed.

[0061] (3) Peptide cleavage

[0062] After the peptide is synthesized, it needs to be cleaved from the resin and the amino protecting groups on the peptide side chains removed to obtain the target peptide. The specific steps are as follows:

[0063] The cleavage reagent (95% TFA / H2O) was slowly added to the resin complex and shaken at room temperature for 3 h;

[0064] Collect the cutting solution into a centrifuge tube, rinse the resin with elution reagent (5% TFA / DCM), and collect the eluate into a centrifuge tube;

[0065] Add an appropriate amount of pre-cooled ether (ether: filtrate = 1:50) into the centrifuge tube, shake thoroughly to mix, and let it stand at low temperature for about 60 minutes to ensure that the polypeptide is fully precipitated;

[0066] Centrifuge at 4000 rpm for 10 min at 4°C, rinse the precipitate with ether, repeat this step three times, and freeze-dry the precipitate in vacuum to obtain the crude polypeptide.

[0067] (4) Purification and identification of peptides

[0068] A small amount of crude polypeptide was taken and purified by preparative high performance liquid chromatography using gradient elution. The peak components were collected, freeze-dried in vacuum, and stored at -20°C for future use.

[0069] 0.1 mg of purified polypeptide was dissolved in 0.5 ml of pure water, filtered through a 0.22 μm filter membrane, and purified using a NanoChrom ChromcoreTM120 C18 (5 μm, 4.6 × 250 mm) reverse phase column with a detection wavelength of 214 nm and a mobile phase of (0.1% TFA / H2O and 0.1% TFA / acetone) at a flow rate of 1 ml / min.

[0070] Another 0.1 mg of purified polypeptide was dissolved in 0.5 ml of pure water, filtered through a 0.22 μm filter membrane, and used for mass spectrometry analysis and identification. Based on the results, the peak of the target product was determined.

[0071] (5) Result determination

[0072] like Figure 3 As shown, the purified MsPiscidin-3 derivative peptide was analyzed by analytical HPLC, and a single elution peak was obtained with a purity greater than 95%. The mass spectrometry results showed that the molecular weight of the synthesized MsPiscidin-3 derivative peptide was 2245.40, which was consistent with the theoretical molecular weight. It can be determined that the synthesized target product is the desired synthesized MsPiscidin-3 derivative peptide, namely F 23 IHHIFNGIANVGRMIHGIV 42 Peptide( Figure 4 ).

[0073] 4. Antibacterial activity analysis and application of largemouth bass Piscidin-3 derivative peptides

[0074] The in vitro antimicrobial activity of MsPiscidin-3-derived peptides was determined using a two-fold dilution method. The selected test strains included Staphylococcus aureus, Escherichia coli, Aeromonas hydrophila, Edwardsiella tarda, Aeromonas veronii, and Vibrio splendidus. These were laboratory-preserved strains of aquatic pathogens that can cause serious infectious diseases in fish and are also important bacterial species that can infect humans. The following operations were performed in a clean bench. The specific steps are as follows:

[0075] (1) Take the bacterial strain stored at -80°C and streak it onto an LB plate, incubate it upside down for 12-18 hours (the culture temperature for Escherichia coli and Staphylococcus aureus is set at 37°C, and the culture temperature for other bacterial strains is set at 28°C);

[0076] (2) A single colony was selected and inoculated into LB culture medium, and cultured at 220 rpm until the logarithmic growth phase (the culture temperature for Escherichia coli and Staphylococcus aureus was set at 37°C, and the culture temperature for other strains was set at 28°C). The absorbance of the bacterial solution at 600 nm (OD600) was measured by UV spectrophotometer, and the culture was diluted to 2 × 105 CFU / ml with LB culture medium;

[0077] (3) Dissolve 1 mg of purified MsPiscidin-3 derivative peptide powder in 1 ml of sterile water to prepare a 1 mg / ml antimicrobial peptide stock solution;

[0078] (4) Add 100 μl of LB culture medium to each well of a sterile 96-well plate. Add 100 μl of antimicrobial peptide stock solution to each well in the first column. After gently pipetting to mix, pipette 100 μl into the wells in the same column in the second column. After mixing, pipette 100 μl into the wells in the next column. Dilute the remaining columns in the same manner as above until the 10th column is mixed and 100 μl of solution is pipetted and discarded.

[0079] (5) 100 μl of the test bacterial solution was added to each well of the 1st to 11th columns of the 96-well plate. The final bacterial solution concentration in each well was 1×105 CFU / ml. The concentrations of antimicrobial drugs in columns 1 to 10 were 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31.25 μg / ml, 15.63 μg / ml, 7.81 μg / ml, 3.91 μg / ml, 1.95 μg / ml, 0.98 μg / ml and 0.49 μg / ml, respectively. 100 μl of LB culture medium was added to each well of column 12. Columns 11 and 12 served as controls.

[0080] (6) The 96-well plate was placed in a constant temperature shaker and cultured at 80 rpm for 18-24 h (the culture temperature for Escherichia coli and Staphylococcus aureus was set to 37°C, and the culture temperature for other bacterial species was set to 28°C). The absorbance of each well was measured by a microplate reader (the detection wavelength for Staphylococcus aureus was 450 nm, and the detection wavelength for other bacterial species was 600 nm).

[0081] The mean of the drug concentration in the wells with no bacterial growth and the adjacent wells with bacterial growth was determined as the MIC of the MsPiscidin-3 derivative peptide for this strain. Each bacterium was repeated three times in parallel, and the entire antibacterial activity experiment was repeated three times to minimize experimental errors.

[0082] (7) Results

[0083] The MsPiscidin-3 derivative peptide has a strong inhibitory effect on Aeromonas hydrophila and also has a certain inhibitory effect on Vibrio splendens; the inhibitory effect on Staphylococcus aureus, Edwardsiella tarda, etc. is weaker (Table 2).

[0084] Table 2 Antibacterial activity analysis of Piscidin-3 derived peptides from largemouth bass

[0085]

[0086] The results in Table 2 show that the MsPiscidin-3 derivative peptide has strong specific antibacterial activity and is expected to be used in antibacterial drugs to replace antibiotics for the prevention and treatment of bacterial diseases such as hemorrhagic disease and septicemia caused by Aeromonas hydrophila infection, and ulcer disease caused by Vibrio splendens, thereby avoiding symptoms such as skin rot and ulcers, visceral enlargement and congestion, and bleeding in fish, providing a safe and environmentally friendly source of disease prevention and control for largemouth bass farming.

Claims

1. An antimicrobial peptide, characterized in that The sequence of the antimicrobial peptide is: FIHHIFNGIANVGRMIHGIV.

2. The method for synthesizing the antimicrobial peptide according to claim 1, wherein include: The steps include obtaining the full-length cDNA of the Piscidin gene, designing the antimicrobial peptide, and synthesizing the antimicrobial peptide.

3. The method for synthesizing the antimicrobial peptide according to claim 2, wherein: The antimicrobial peptide design steps include: predicting the N-terminal signal peptide based on the MsPiscidin-3 amino acid sequence using SignalP 5.0, predicting the protein secondary structure using PSIPRED 4.0, predicting the protein physicochemical properties using the ProtParam tool, analyzing the molecular weight, isoelectric point, fat coefficient, instability coefficient and hydrophilicity coefficient, and designing and modifying MsPiscidin-3 based on protein secondary structure simulation, focusing on key elements such as the signal peptide, antimicrobial 12 domains and pro-protease cleavage site, and determining the FIHHIFNGIANVGRMIHGIV sequence as the MsPiscidin-3 derivative peptide.

4. The method for synthesizing the antimicrobial peptide according to claim 2, wherein: The step of obtaining the full-length cDNA of the Piscidin gene comprises: cloning and obtaining the full-length cDNA of the largemouth bass antimicrobial peptide Piscidin gene by using RT-PCR and RACE-PCR methods.

5. The method for synthesizing the antimicrobial peptide according to claim 4, characterized in that: The step of obtaining the full-length cDNA of the Piscidin gene specifically comprises: taking spleen tissue of largemouth bass, extracting total RNA by using the Trizol method, using the total RNA as a template, and synthesizing the first-strand cDNA of largemouth bass, 5'RACE cDNA and 3'RACE cDNA by using a reverse transcription kit and a RACE cDNA amplification kit respectively; Using the first strand of cDNA as a template, the middle fragment sequence of the Piscidin gene of largemouth bass was obtained through PCR amplification, product recovery and sequencing. 5'RACE and 3'RACE primers were designed based on the sequencing results of the intermediate fragments, and 5'RAC cDNAE and 3'RACE cDNA were used as templates, respectively. PCR amplification, product recovery, and sequencing were also performed to obtain 5'RACE and 3'RACE gene fragments. The sequence results obtained by sequencing were spliced ​​using Seqman software, and the corresponding sequences of other species in the database were searched, aligned, and analyzed using the BLAST service of NCBI; The corresponding amino acid sequences were deduced using the translation tool on the ExPASy website. The amino acid sequence homology between different species was analyzed using MatGAT2.02 software. Protein domains were predicted using the SMART online tool and the conserved domain prediction service of NCBI. A phylogenetic tree was constructed using the neighbor-joining method using MEGA 11 software, with a bootstrapping value of 10,000.

6. The method for synthesizing the antimicrobial peptide according to claim 2, characterized in that: The step of synthesizing the antimicrobial peptide comprises: synthesizing a MsPiscidin-3 derivative peptide as the antimicrobial peptide by using an Fmoc solid phase synthesis method.

7. The method for synthesizing the antimicrobial peptide according to claim 6, characterized in that: The steps of synthesizing the antimicrobial peptide include: activation of resin, condensation of amino acids, cleavage of polypeptide, and purification and identification of polypeptide.

8. Use of the antimicrobial peptide according to claim 1 in the preparation of an antimicrobial drug for largemouth bass, characterized in that: The bacteria targeted by the antibacterial drug are Aeromonas hydrophila or Vibrio splendens.

9. Use of the antimicrobial peptide according to claim 1 in the preparation of an antimicrobial drug for preventing and treating bacterial diseases of largemouth bass such as hemorrhagic disease and septicemia caused by Aeromonas hydrophila infection and ulcerous disease caused by Vibrio splendens.

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