Antibacterial peptide as well as synthesis method and application thereof

Abstract

The invention discloses an antibacterial peptide as well as a synthesis method and application thereof, and belongs to the field of antibacterial peptides. The sequence of the antibacterial peptide is as follows: F23IHHIFNGIANVGRMIHGIV42, and the sequence of the antibacterial peptide is as follows. The synthetic method of the antibacterial peptide comprises the following steps: obtaining the full-length cDNA of the Piscidin gene, designing the antibacterial peptide, and synthesizing the antibacterial peptide. The micropterus salmoides strain is applied to the aspect of micropterus salmoides antibiosis, solves the problem that the development of the micropterus salmoides culture industry is restricted due to drug resistance and water environment pollution caused by the fact that antibiotic drugs are adopted by the existing micropterus salmoides to prevent diseases, and has a relatively strong specific immune function on aeromonas hydrophila and vibrio splendidus.

Description

Technical Field

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

[0002] Antimicrobial peptides (AMPs), also known as antimicrobial peptides and host defense peptides (HDPs), are a class of small bioactive polypeptides widely present in nature and are also an important part of the body's innate immunity. Antimicrobial peptides have broad-spectrum antibacterial activity and have strong inhibitory effects on Gram-negative bacteria, Gram-positive bacteria, fungi, etc. Some antimicrobial peptides also have anti-tumor activity, can regulate inflammatory responses, and have the characteristics of being pollution-free, residue-free, broad-spectrum antibacterial and not easily developing drug resistance. They are known as "natural antibiotics" and are expected to replace antibiotics for the prevention and control of pathogenic diseases in aquatic animals.

[0003] Antimicrobial peptides are mainly obtained in three ways: extracting endogenous antimicrobial peptides from organisms, chemical synthesis, and preparation by genetic engineering expression. To date, the antimicrobial peptides found in fish mainly include Piscidins, β-defensins, Hepcidins, Cathelicidins, liver-expressed antimicrobial peptides 2 (LEAP-2), and NK-lysins. Among them, Piscidins are one of the most common antimicrobial peptides in fish and are further divided into subtypes such as Pleurocidins, Misgurins, Piscidins, Moronecidins, Epinecidins, Dicentracins, and Gaduscidins. These subtype peptides usually consist of 18-27 amino acid residues, have an α-helical structure, belong to the same evolutionarily related family, but have relatively low sequence homology. According to sequence similarity and phylogenetic analysis, Piscidin subtypes are further divided into three categories: Piscidin-1, Piscidin-2, and Piscidin-3. Their main differences are as follows: The Piscidin-1 sequence is longer than those of 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 mature peptide C-terminus of Piscidin-2 has an H-R / K proprotein convertase cleavage motif, while the cleavage motif of Piscidin-3 is R-R-R-H.

[0004] Micropterus salmoides is one of the main varieties of freshwater aquaculture in China. With the expansion of the aquaculture scale and the improvement of intensification, bacterial and viral diseases frequently occur during the growth process. Currently, antibiotic drugs are mainly used to prevent diseases. Long-term use will produce varying degrees of drug resistance, causing economic losses and polluting the water environment, seriously restricting the development of the Micropterus salmoides aquaculture industry. There is little research on the immune response of Micropterus salmoides after being infected by bacteria and viruses. The molecular level and mechanism of action of Piscidins antibacterial peptides are still blank, greatly restricting the technical development of preventing diseases by immunological methods. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the technical problem to be solved by the present invention is to overcome the problems that the existing Micropterus salmoides uses antibiotic drugs to prevent diseases, resulting in drug resistance and water environment pollution, thus restricting the development of the Micropterus salmoides aquaculture industry. The present invention proposes an antibacterial peptide with strong specific immune function against Aeromonas hydrophila and Vibrio splendidus, its synthesis method and application.

[0006] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] On the one hand, the present invention provides an antibacterial peptide, and the sequence of the antibacterial peptide is: F 23 IHHIFNGIANVGRMIHGIV 42 。

[0008] On the other hand, the present invention provides a synthesis method of the above antibacterial peptide, including: the step of obtaining the full-length cDNA of the Piscidin gene, the step of designing the antibacterial peptide, and the step of synthesizing the antibacterial peptide.

[0009] Preferably, the step of designing the antibacterial peptide includes: based on the amino acid sequence of MsPiscidin-3, using SignalP5.0 to predict the N-terminal signal peptide, predicting the secondary structure of the protein by PSIPRED 4.0, using the ProtParam tool to predict the physicochemical properties of the protein, analyzing the molecular weight, isoelectric point, aliphatic coefficient, instability coefficient and hydrophilicity coefficient, and according to the simulation of the protein secondary structure, focusing on several key elements such as the signal peptide, antibacterial 12 domain and preproprotein cleavage site, designing and modifying MsPiscidin-3 to determine F 23 IHHIFNGIANVGRMIHGIV 42 sequence as the MsPiscidin-3 extended peptide.

[0010] Preferably, the step of obtaining the full-length cDNA of the Piscidin gene includes: cloning the full-length cDNA of the Micropterus salmoides antibacterial peptide Piscidin gene by using RT-PCR and RACE-PCR methods.

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

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

[0013] Design 5' RACE and 3' RACE primers according to the sequencing results of the middle fragment, respectively using 5' RAC cDNA E and 3' RACE cDNA as templates, and also through PCR amplification, product recovery, and sequencing methods to obtain 5' RACE and 3' RACE gene fragments;

[0014] Splice the sequence results obtained by sequencing using Seqman software, and search, align, and analyze the corresponding sequences of other species in the database through the BLAST service of NCBI;

[0015] Use the translation tool on the ExPASy website to deduce the corresponding amino acid sequence; use the MatGAT 2.02 software to analyze the amino acid sequence homology among different species; use the SMART online tool and the conserved domain prediction service of NCBI to predict protein domains; use the MEGA 11 software to construct a phylogenetic tree based on the neighbor-joining method, with a Bootstrap value of 10,000 times.

[0016] Preferably, the steps for synthesizing the antimicrobial peptide include: synthesizing the MsPiscidin-3 derivative peptide using the Fmoc solid-phase synthesis method as the antimicrobial peptide.

[0017] Preferably, the steps for synthesizing the antimicrobial peptide successively include: 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 above antimicrobial peptide in the antibacterial aspect of the largemouth bass.

[0019] Preferably, the antimicrobial peptide is used as an antibacterial drug or a feed additive for antibacterial of the largemouth bass.

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

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

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

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

[0024] Figure 2 is the phylogenetic analysis of Piscidins in different teleost fishes. Among them, blue, red, and green represent Piscidins-1, Piscidins-2, and Piscidins-3 respectively; the gene accession numbers are listed after the species names;

[0025] Figure 3 is the HPLC analysis of the Piscidin-3-derived peptide of largemouth bass;

[0026] Figure 4 is the mass spectrometry analysis of the Piscidin-3-derived peptide of largemouth bass. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The technical solutions in the specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific embodiments of the overall technical solution of the present invention, rather than all embodiments. Based on the overall concept of the present invention, all other embodiments obtained by those of ordinary skill in the art fall within the protection scope of the present invention.

[0028] On the one hand, the present invention provides an antibacterial peptide, and the sequence of the antibacterial peptide is: F 23 IHHIFNGIANVGRMIHGIV 42。The fat coefficient of this antimicrobial peptide is 131.50, and the instability index is 14.13. Its stability is much higher than that of the original peptide, making it easy to be synthesized subsequently and used for subsequent activity analysis and applications. This antimicrobial peptide has a strong inhibitory effect on Aeromonas hydrophila and also has a certain inhibitory effect on Vibrio splendidus, indicating that the MsPiscidin-3-derived peptide has strong specific antibacterial activity and is expected to be applied to antibacterial drugs and feed additives.

[0029] On the other hand, the present invention provides a method for synthesizing the above-mentioned antimicrobial peptide, including: the step of obtaining the full-length cDNA of the Piscidin gene, the step of designing the antimicrobial peptide, and the step of synthesizing the antimicrobial peptide.

[0030] Among them, the step of designing the antimicrobial peptide includes: based on the amino acid sequence of MsPiscidin-3, using SignalP 5.0 to predict the N-terminal signal peptide, predicting the protein secondary structure through PSIPRED 4.0, using the ProtParam tool to predict the physicochemical properties of the protein, analyzing the molecular weight, isoelectric point, fat coefficient, instability coefficient and hydrophilicity coefficient, and according to the protein secondary structure simulation, focusing on several key elements such as the signal peptide, antibacterial 12 domain and preproprotein cleavage site, designing and modifying MsPiscidin-3 to determine the F 23 IHHIFNGIANVGRMIHGIV 42 sequence as the MsPiscidin-3 extended peptide.

[0031] Furthermore, the steps for obtaining the full-length cDNA of the Piscidin gene include: cloning the full-length cDNA of the antimicrobial peptide Piscidin gene from the largemouth bass using RT-PCR and RACE-PCR methods. Specifically: taking the spleen tissue of the largemouth bass, extracting total RNA using the Trizol method, using this as a template, and respectively using a reverse transcription kit and a RACE cDNA amplification kit to synthesize the first strand of largemouth bass cDNA, 5'RACE, and 3'RACE cDNA; using the first strand of cDNA as a template, obtaining the middle fragment sequence of the Piscidin gene of the largemouth bass through PCR amplification, product recovery, and sequencing; designing 5'RACE and 3'RACE primers according to the sequencing results of the middle fragment, and respectively using 5'RAC cDNA E and 3'RACE cDNA as templates, and also obtaining 5'RACE and 3'RACE gene fragments through PCR amplification, product recovery, and sequencing methods; splicing the sequence results obtained by sequencing using Seqman software, and searching, aligning, and analyzing the corresponding sequences of other species in the database through the BLAST service of NCBI; using the translation tool on the ExPASy website to deduce the corresponding amino acid sequence; using the MatGAT 2.02 software to analyze the amino acid sequence homology among different species; using the SMART online tool and the conserved domain prediction service of NCBI to predict the protein domain; using the MEGA11 software to construct a phylogenetic tree based on the neighbor-joining method, with the Bootsraps value being 10,000 times.

[0032] In a preferred embodiment, the steps for synthesizing the antimicrobial peptide include: synthesizing the MsPiscidin-3 derivative peptide as the antimicrobial peptide using the Fmoc solid-phase synthesis method. It sequentially includes: activation of the resin, condensation of amino acids, cleavage of the polypeptide, and purification and identification of the polypeptide.

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

[0034] The present invention also provides the application of the above-mentioned antimicrobial peptide in antibacterial of largemouth bass. Specifically, the antimicrobial peptide is used as an antibacterial drug or feed additive for antibacterial of largemouth bass. Further, the antimicrobial peptide is used as an antibacterial drug or feed additive for preventing and treating hemorrhagic disease and septicemia of largemouth bass caused by Aeromonas hydrophila infection, and ulcerous bacterial diseases of largemouth bass caused by Vibrio splendidus. It should be noted that the above-mentioned MsPiscidin-3-derived peptide has a strong inhibitory effect on Aeromonas hydrophila and also has a certain inhibitory effect on Vibrio splendidus; it has a weak inhibitory effect on Staphylococcus aureus, Edwardsiella tarda, etc., indicating that the MsPiscidin-3-derived peptide has strong specific antibacterial activity and is expected to be applied to antibacterial drugs and feed additives to replace antibiotics for preventing and treating hemorrhagic disease and septicemia caused by Aeromonas hydrophila infection, and ulcerous diseases caused by Vibrio splendidus and other bacterial diseases, avoiding symptoms such as skin rot and ulceration, visceral swelling and congestion, and bleeding of fish bodies, providing a safe and environmentally friendly source for disease prevention and control in largemouth bass farming, and having the advantages of safety, high efficiency, no pollution, and environmental protection.

[0035] In order to introduce the antimicrobial peptide, its synthesis method and application provided by the embodiments of the present invention more clearly and in detail, the following will be described in combination with specific embodiments.

[0036] Example

[0037] Preparation of experimental animals: Healthy largemouth bass (body weight 50.0 ± 5.0 g) were temporarily raised in a laboratory recirculating water aquaculture system. During this period, the water temperature was maintained at 25.0 ± 3.0 °C, the light cycle was 14 h light / 10 h darkness, and they were fed regularly, quantitatively and at fixed points twice a day.

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

[0039] (1) Gene cloning steps

[0040] The full-length cDNA of the antimicrobial peptide Piscidin gene of largemouth bass was cloned by RT-PCR and RACE-PCR methods. Specifically, largemouth bass spleen tissue was taken, and total RNA was extracted by the Trizol method. Using this as a template, the first strand of largemouth bass cDNA, 5'RACE and 3'RACE cDNA were synthesized 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 largemouth bass Piscidin gene was obtained through PCR amplification, product recovery, and sequencing. According to the sequencing results of the middle fragment, 5'RACE and 3'RACE primers were designed, and using 5'RAC cDNA E and 3'RACE cDNA as templates respectively, 5'RACE and 3'RACE gene fragments were obtained through the same methods of PCR amplification, product recovery, and sequencing.

[0041] (2) Sequence analysis process

[0042] The sequence results obtained by sequencing were assembled using Seqman software, and the corresponding sequences of other species in the database were searched, aligned, and analyzed through the BLAST service of NCBI. The corresponding amino acid sequences were deduced using the translation tool on the ExPASy website. The MatGAT 2.02 software was used to analyze the amino acid sequence homology among different species. The SMART online tool and the conserved domain prediction service of NCBI were used to predict protein domains. The MEGA 11 software was used to construct a phylogenetic tree based on the neighbor-joining method (N-J), with a Bootstrap value of 10,000 times.

[0043] (3) Results

[0044] As Figure 1 shown, the full-length Piscidin cDNA sequence of Micropterus salmoides is 401 bp, encoding 63 amino acids. As Figure 2 shown, according to the phylogenetic tree analysis, the cloned antimicrobial peptide gene was classified into the Piscidin-3 class and named MsPiscidin-3. Through domain prediction, it was found that MsPiscidin-3 has an antimicrobial 12 domain. Research has shown that the antimicrobial 12 domain mainly uses its amphiphilic α-helical structure to pierce the bacterial cell membrane and plays a crucial role in antimicrobial activity.

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

[0046] Based on the translated MsPiscidin-3 amino acid sequence, 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 physicochemical properties of the protein, analyzing parameters such as molecular weight, isoelectric point, aliphatic index, instability index, and hydrophilicity coefficient to determine the synthesis target. Based on the amino acid sequence and structural characteristics of MsPiscidin-3, MsPiscidin-3-derived peptides were designed. According to the protein secondary structure simulation, key elements such as the signal peptide, antimicrobial 12 domain, and proprotein convertase cleavage site were mainly considered for the design and modification of MsPiscidin-3, and the sequence 23 F 42 IHHIFNGIANVGRMIHGIV was selected as the MsPiscidin-3-derived peptide. As shown in Table 1, the aliphatic index of the derived peptide is 131.50, and the instability index is 14.13, with much higher stability than the original peptide, making it easy for subsequent synthesis and for use in subsequent activity analysis and applications.

[0047] Table 1 Comparison of Physicochemical Characteristics between Micropterus salmoides Piscidin-3 and Its Derived Peptides

[0048]

[0049]

[0050] 3. Solid-phase Synthesis of Micropterus salmoides Piscidin-3-derived Peptides

[0051] MsPiscidin-3-derived peptides were synthesized on a Tetras peptide synthesizer using the Fmoc solid-phase synthesis method and purified by preparative HPLC. The specific steps are as follows:

[0052] (1) Activation of Resin

[0053] Swelling of resin: Take 1 g of Fmoc-Rink-Amide-AM resin and place it in a solid-phase reactor. Wash it with DCM, filter by suction, add 10 ml of DCM and soak for 30 min to fully swell the resin, then filter by suction to remove the excess solvent;

[0054] Removal of Fmoc protecting group: Add 10 ml of 20% piperidine / DMF to wash, filter by suction; add 10 ml of 20% piperidine / DMF to wash, shake and mix evenly, stir and react for 30 min; drain, add DMF, isopropanol and DMF in sequence and shake and rinse thoroughly several times, then drain;

[0055] Ninhydrin test (Kaiser test): Dip a small amount of resin into a small tube, dropwise add ninhydrin / ethanol, phenol / ethanol and KCN / pyridine in sequence, heat in a boiling water bath. If the resin particles turn blue-violet, it indicates successful deprotection.

[0056] (2) Condensation of Amino Acids

[0057] Take Fmoc-AA-OH and HOBt and dissolve them in DMF, shake and mix evenly, then add DCC and shake and mix evenly;

[0058] Add the above solution to the solid-phase reactor and react with shaking at room temperature for 3 h;

[0059] Rinse several times with DMF, isopropanol and DMF in sequence, then drain;

[0060] Perform the ninhydrin test according to the method in (1): If the resin particles do not show color, it indicates that the condensation is completed; if the resin shows color, repeat the process.

[0061] Remove the Fmoc protecting group according to the method in (1), and add amino acids in sequence according to the sequence of MsPiscidin-3-derived peptides, and repeat the above process until all amino acids are successfully condensed.

[0062] (3) Cleavage of Polypeptide

[0063] After the polypeptide synthesis is completed, it is necessary to cleave it from the resin and remove the amino protecting groups on the side chains of the peptide chain to obtain the target peptide. The specific steps are as follows:

[0064] Slowly add the cleavage reagent (95% TFA / H2O) to the resin complex and react with shaking at room temperature for 3 h;

[0065] Collect the cleavage solution into a centrifuge tube, and rinse the resin with the elution reagent (5% TFA / DCM), and collect the eluate into the centrifuge tube together;

[0066] Add an appropriate amount of pre-cooled ether (ether: filtrate = 1:50) to the centrifuge tube, shake well, and let it stand at low temperature for about 60 min to ensure that the polypeptide precipitates fully;

[0067] Centrifuge at 4 °C and 4000 rpm for 10 min, rinse the precipitate with ether, repeat this step three times, and freeze-dry the precipitate under vacuum to obtain the crude polypeptide.

[0068] (4) Purification and identification of the polypeptide

[0069] Take a small amount of the crude polypeptide and purify the polypeptide using a preparative high-performance liquid chromatograph. Use gradient elution, collect the components of each peak, freeze-dry them under vacuum, and store them at -20 °C for later use.

[0070] Take 0.1 mg of the purified polypeptide and dissolve it in 0.5 ml of pure water, filter it through a 0.22 μm filter membrane, use a NanoChrom ChromcoreTM120 C18 (5 μm, 4.6 × 250 mm) reversed-phase column, detection wavelength: 214 nm, mobile phase: (0.1% TFA / H 2 O and 0.1% TFA / acetone), flow rate: 1 ml / min.

[0071] Take another 0.1 mg of the purified polypeptide and dissolve it in 0.5 ml of pure water, filter it through a 0.22 μm filter membrane for mass spectrometry analysis and identification. According to the results, determine the peak where the target product is located.

[0072] (5) Result determination

[0073] As Figure 3 shown, the MsPiscidin-3-derived peptide obtained by purification was analyzed by analytical HPLC, and a single elution peak was obtained, with a purity greater than 95%. The results of mass spectrometry detection showed that the molecular weight of the purified MsPiscidin-3-derived peptide was 2245.40, which was consistent with the theoretical molecular weight. It can be determined that the synthesized target product is the MsPiscidin-3-derived peptide to be synthesized, that is, F 23 IHHIFNGIANVGRMIHGIV 42 peptide segment(Figure 4 )。

[0074] 4. Antibacterial Activity Analysis and Application of Micropterus salmoides Piscidin-3 Derived Peptide

[0075] The in vitro antibacterial activity of MsPiscidin-3 derived peptide was determined by the two-fold dilution method. The selected test bacterial strains included Staphylococcus aureus, Escherichia coli, Aeromonas hydrophila, Edwardsiella tarda, Aeromonas veronii, and Vibrio splendidus. These were strains preserved in the laboratory, all of which were aquatic pathogens that could cause severe infectious diseases in fish and important strains that could infect humans. The following operations were all carried out in a laminar flow hood, and the specific steps were as follows:

[0076] (1) Streak inoculate the strains stored at -80°C on LB plates and incubate them upside down for 12 - 18 h (the culture temperature for Escherichia coli and Staphylococcus aureus was set at 37°C, and the culture temperature for the remaining strains was set at 28°C);

[0077] (2) Pick a single colony and inoculate it into LB culture medium, and shake it 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 the remaining strains was set at 28°C). Use an ultraviolet spectrophotometer to detect the absorbance value (OD600) of the bacterial solution at 600 nm, and dilute it to 2×105 CFU / ml with LB culture medium;

[0078] (3) Dissolve 1 mg of purified MsPiscidin-3 derived peptide dry powder in 1 ml of sterile water to prepare an antibacterial peptide stock solution of 1 mg / ml;

[0079] (4) Add 100 μl of LB culture medium to each well of a sterile 96-well plate. Add 100 μl of the antibacterial peptide stock solution to each well in the first column. After gently aspirating and mixing, pipette 100 μl and add it to the wells in the same row of the second column. After mixing, pipette 100 μl and add it to the wells in the same row of the next column. Dilute the remaining columns in the same way as above until 100 μl of the solution is aspirated and discarded after mixing in the tenth column;

[0080] (5) Add 100 μl of the bacterial solution to be tested to each well in columns 1 to 11 of the 96-well plate. The final concentration of the bacterial solution in each well is 1×105 CFU / ml. The concentrations of the antibacterial drugs in columns 1 to 10 are 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 in sequence. Add 100 μl of LB culture medium to each well in column 12. Columns 11 and 12 serve as controls.

[0081] (6) Place the 96-well plate in a constant temperature shaker and shake it at 80 rpm for 18 - 24 h (the culture temperature for Escherichia coli and Staphylococcus aureus is set at 37 °C, and the culture temperature for the remaining bacterial strains is set at 28 °C). Measure the absorbance value of each well with an enzyme-linked immunosorbent assay (ELISA) reader (the detection wavelength for Staphylococcus aureus is 450 nm, and the detection wavelengths for the remaining bacterial strains are all 600 nm).

[0082] The average value of the drug concentration in the well where no bacterial growth is detected and the drug concentration in the adjacent well where bacterial growth is detected is determined as the MIC of the MsPiscidin-3-derived peptide against this bacterial strain. Each bacterium is repeated in parallel three times, and the entire antibacterial activity experiment is repeated three times to minimize experimental errors.

[0083] (7) Results

[0084] The MsPiscidin-3-derived peptide has a strong inhibitory effect on Aeromonas hydrophila and also has a certain inhibitory effect on Vibrio splendidus; its inhibitory effect on Staphylococcus aureus, Edwardsiella tarda, etc. is relatively weak (Table 2).

[0085] Table 2 Analysis of the antibacterial activity of the Piscidin-3-derived peptide from Micropterus salmoides

[0086]

[0087] The results in Table 2 show that the MsPiscidin-3-derived peptide has strong specific antibacterial activity and is expected to be applied to antibacterial drugs and feed additives to replace antibiotics for the prevention and treatment of hemorrhagic diseases and septicemia caused by Aeromonas hydrophila infection, as well as bacterial diseases such as ulcer disease caused by Vibrio splendidus, avoiding symptoms such as skin rot and ulceration, visceral enlargement and congestion, and bleeding in fish, providing a safe and environmentally friendly source for disease prevention and control in Micropterus salmoides farming.

Claims

1. An antimicrobial peptide, characterized in that: The sequence of the antimicrobial peptide is: 23 IHHIFNGIANVGRMIHGIV 42 .

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

3. The method for synthesizing the antimicrobial peptide according to claim 2, characterized in that: The antimicrobial peptide design steps include: predicting the N-terminal signal peptide based on the amino acid sequence of MsPiscidin-3 using SignalP 5.0, predicting the protein secondary structure using PSIPRED 4.0, predicting the physicochemical properties of the protein 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 of signal peptide, antimicrobial 12 domain and pro-protease cleavage site, and determining the F 23 IHHIFNGIANVGRMIHGIV 42 The sequence is used as the MsPiscidin-3 prolonging peptide.

4. The method for synthesizing the antimicrobial peptide according to claim 2, characterized in that: The step of obtaining the full-length cDNA of the Piscidin gene comprises: cloning and obtaining the full-length cDNA of the antibacterial peptide Piscidin gene of largemouth bass 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 includes: taking spleen tissue of a largemouth bass, extracting total RNA by using the Trizol method, using the total RNA as a template, and synthesizing the first-chain cDNA of the largemouth bass, 5'RACE 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. According to the results of intermediate fragment sequencing, 5'RACE and 3'RACE primers were designed, and 5'RACcDNA E and 3'RACE cDNA were used as templates, respectively. 5'RACE and 3'RACE gene fragments were obtained by PCR amplification, product recovery, and sequencing. 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 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. The protein domains were predicted using the SMART online tool and the conserved domain prediction service of NCBI. The phylogenetic tree was constructed using MEGA 11 software based on the neighbor-joining method, with a Bootsraps value of 10,000 times.

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 the 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 peptides sequentially include: activation of resin, condensation of amino acids, cleavage of polypeptides, and purification and identification of polypeptides.

8. Use of the antimicrobial peptide according to claim 1 in antibacterial treatment of largemouth bass.

9. The use of the antimicrobial peptide in antibacterial treatment of largemouth bass according to claim 8, characterized in that: The antimicrobial peptide is used as an antimicrobial drug or a feed additive for antibacterial treatment of largemouth bass.

10. The use of the antimicrobial peptide in antibacterial treatment of largemouth bass according to claim 8, characterized in that: The antimicrobial peptide is used as an antimicrobial drug or feed additive to prevent and treat hemorrhagic disease and septicemia of largemouth bass caused by Aeromonas hydrophila infection, and ulcerous bacterial diseases of largemouth bass caused by Vibrio splendens.

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