Improved Mytichitin-CB antimicrobial peptide and its encoding gene, recombinant plasmid, recombinant bacteria and applications

Abstract

This invention discloses a modified Mytichitin-CB antimicrobial peptide, its encoding gene, recombinant plasmid, recombinant bacteria, and its applications. The modified Mytichitin-CB antimicrobial peptide is either a first antimicrobial peptide or a second antimicrobial peptide; the amino acid sequence of the first antimicrobial peptide is shown in SEQ ID NO.3; the amino acid sequence of the second antimicrobial peptide is shown in SEQ ID NO.5. The modified peptide of this invention is obtained by targeted molecular modification of the antimicrobial peptide Mytichitin-CB using genetic engineering and site-directed mutagenesis techniques, starting from the charge number and amphiphilicity of the antimicrobial peptide. Compared with the original antimicrobial peptide Mytichitin-CB, the modified peptide of this invention has significantly improved antibacterial and bactericidal effects, exhibits high stability in different pH and temperature environments, and has low cytotoxicity, making it suitable for widespread application in livestock and poultry farming, medicine, and other fields.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and more specifically, to the modified Mytichitin-CB antimicrobial peptide and its encoding gene, recombinant plasmid, recombinant bacteria, and applications. Background Technology

[0002] Antimicrobial peptides (AMPs) have attracted attention and in-depth research due to their broad-spectrum antibacterial activity and unique mechanisms of action, making them a popular alternative to antibiotics. AMPs are polypeptides encoded by ribosomal genes, possessing innate defensive capabilities and novel abilities against multidrug-resistant bacterial pathogens. They are typically small peptides composed of 20-60 amino acids, exhibiting strong cationicity (pI 8.9–10.7), thermal stability (100℃, 15 minutes), and no drug fastness. These properties make antimicrobial peptides promising antibacterial agents.

[0003] Antimicrobial peptides are widely expressed in organisms and are associated with the innate immunity of invertebrates. When marine mollusks are invaded by microorganisms or suffer damage, they secrete non-specific immune factors such as antimicrobial peptides to defend against and kill invading substances. Marine fish-derived antimicrobial peptides have antimicrobial functions, including a certain killing effect on pathogens such as Gram-positive bacteria, Gram-negative bacteria, fungi, and viruses. Mytichitin-CB is a novel antimicrobial peptide with 55 amino acid residues and a molecular weight of 6621.55 Da, isolated from marine thick-shelled mussels by Qin et al. in 2014. It is a novel peptide rich in cysteine, disulfide bonds, and chitin-binding domains. Antimicrobial experiments showed that Mytichitin-CB exhibited antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria, and fungi, and also had antiparasitic activity.

[0004] Previously, the laboratory successfully obtained a recombinant antimicrobial peptide, Mytichitin-CB, with relatively high yield, good activity, and stability through genetic engineering. For example, patent application CN 110105441 A obtained a recombinant Mytichitin-CB antimicrobial peptide by adding "GATGACGATGACAAG" to the 5' end of the Mytichitin-CB target gene. This antimicrobial peptide showed strong inhibitory effects against Staphylococcus aureus, Listeria monocytogenes, Pseudomonas fluorescens, Escherichia coli, Salmonella, Bacillus subtilis L300-1, and Enterobacter aerogenes, but the minimum inhibitory concentration was around 6 μg / mL. Therefore, it is necessary to provide a modified antimicrobial peptide with better antibacterial effects. Summary of the Invention

[0005] The present invention provides a modified Mytichitin-CB antimicrobial peptide, which is a first antimicrobial peptide or a second antimicrobial peptide; the amino acid sequence of the first antimicrobial peptide is shown in SEQ ID NO.3; the amino acid sequence of the second antimicrobial peptide is shown in SEQ ID NO.5.

[0006] The present invention provides the encoding gene of the aforementioned modified Mytichitin-CB antimicrobial peptide, which is a first gene or a second gene; the first gene is used to encode the first antimicrobial peptide, and the nucleotide sequence is shown in SEQ ID NO.15; the second gene is used to encode the second antimicrobial peptide, and the nucleotide sequence is shown in SEQ ID NO.16.

[0007] The present invention provides a recombinant plasmid encoding the aforementioned gene, which is a first recombinant plasmid or a second recombinant plasmid; the first recombinant plasmid includes the first gene; the second recombinant plasmid includes the second gene; and the expression plasmid of the first recombinant plasmid or the second recombinant plasmid is pPICZαA, respectively.

[0008] The present invention provides a recombinant strain of the aforementioned recombinant plasmid, which is a first recombinant strain or a second recombinant strain; the first recombinant strain contains a first recombinant plasmid; the second recombinant strain contains a second recombinant plasmid; the original strain of the recombinant strain is Pichia pastoris.

[0009] Preferably, the Pichia pastoris is Pichia pastoris X-33.

[0010] This invention provides the application of the aforementioned modified Mytichitin-CB antimicrobial peptide, its encoding gene, recombinant plasmid, or recombinant bacteria in the preparation of antibacterial agents.

[0011] Preferably, the bacteria are one or more of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, Enterococcus faecalis, Pseudomonas tatarsus, Enterobacter aerogenes, or Salmonella.

[0012] More preferably, the bacteria are Staphylococcus aureus or Salmonella.

[0013] The beneficial effects of this invention include: Starting from the charge number and amphiphilicity of the antimicrobial peptide Mytichitin-CB, this invention utilizes genetic engineering and site-directed mutagenesis technology to perform targeted molecular modification of the antimicrobial peptide, thereby obtaining the improved peptides rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R. Compared with the original antimicrobial peptide Mytichitin-CB, the improved peptides of this application have significantly improved antibacterial (rMytichitin-CB-D20I-D23R has more than 2 times the antibacterial activity against Staphylococcus aureus; rMytichitin-CB-D23H has a MIC of 0.17 μg / mL against Salmonella, which is significantly lower than the value reported in existing literature) and bactericidal effects. They also exhibit high stability and low cytotoxicity in different pH and temperature environments, making them suitable for widespread application in livestock and poultry farming, medicine, and other fields. Attached Figure Description

[0014] Figure 1 These are PCR amplification verification diagrams of the various modified peptide recombinant expression vectors provided in Example 2 of the present invention. Where M is a 1kb DNA ladder; 1 represents PCR amplification of pPICZαA-rMytichitin-CB-D20I; 2 represents PCR amplification of pPICZαA-rMytichitin-CB-D23H; 3 represents PCR amplification of pPICZαA-rMytichitin-CB-D20I-H39I; and 4 represents PCR amplification of pPICZαA-rMytichitin-CB-D20I-D23R.

[0015] Figure 2 This is a double enzyme digestion verification diagram of the target gene and expression vector provided in Example 2 of the present invention, where M is a 100bp DNA ladder; 1 is the EcoRI and KpnI double enzyme digestion plasmid pPICZαA-rMytichitin-CB-D20I; 2 is the EcoRI and KpnI double enzyme digestion plasmid pPICZαA-rMytichitin-CB-D23H; 3 is the EcoRI and KpnI double enzyme digestion plasmid pPICZαA-rMytichitin-CB-D20I-H39I; and 4 is the EcoRI and KpnI double enzyme digestion plasmid pPICZαA-rMytichitin-CB-D20I-D23R.

[0016] Figure 3The diagram shows the bactericidal kinetics of rMytichitin-CB and three modified peptides against Staphylococcus aureus provided in Example 3 of this invention; where ad represents the bactericidal kinetics of Mytichitin-CB, rMytichitin-CB-D20I, rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R against Staphylococcus aureus, respectively.

[0017] Figure 4 The diagram shows the bactericidal kinetics of Mytichitin-CB and three modified peptides against Salmonella provided in Example 3 of this invention; where ad represents the bactericidal kinetics of Mytichitin-CB, rMytichitin-CB-D20I, rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R against Salmonella, respectively.

[0018] Figure 5 The effect of different temperatures on the stability of Mytichitin-CB and four modified peptides provided in Example 3 of the present invention; wherein the test bacteria in a is Staphylococcus aureus and the test bacteria in b is Salmonella.

[0019] Figure 6 The effect of different pH environments provided in Example 3 of the present invention on the stability of Mytichitin-CB and four modified peptides, wherein the test bacteria in a is Staphylococcus aureus and the test bacteria in b is Salmonella.

[0020] Figure 7 This invention illustrates the effects of different proteases provided in Example 3 on the stability of Mytichitin-CB and four modified peptides. rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R exhibit certain protease stability (papain, pepsin, proteinase K, or trypsin). Figure a shows the test bacteria as Staphylococcus aureus, and Figure b shows Salmonella; the vertical axis represents the inhibition rate (%), and the horizontal axis represents different proteases.

[0021] Figure 8 The cytotoxicity of rMytichitin-CB and four modified peptides to mammalian cells provided in Example 3 of the invention is shown in Figure 3, wherein a is bovine kidney cells (MDBK), b is Coxsbane dog kidney cells (MDCK), c is baby hamster kidney cells (BHK 21), and d is African green monkey kidney cells (Vero). Detailed Implementation

[0022] The present invention will be further described and illustrated below with reference to embodiments. However, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the present invention and the embodiments, all other inventions and embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] Example 1: Improved peptide molecular design

[0026] Increasing the number of positive charges can not only improve the binding ability of antimicrobial peptides to bacterial cell membranes, but also effectively reduce the bactericidal concentration of antimicrobial peptides. Based on the number of charges, we first replaced the negatively charged amino acid Asp at position 23 of Mytichitin-CB (its amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.6) with the positively charged His, adding 2 positive charges to obtain the rMytichitin-CB-D23H modified peptide (its amino acid sequence is shown in SEQ ID NO.3).

[0027] Hydrophobicity is an important factor affecting the antimicrobial activity of antimicrobial peptides. For antimicrobial peptides with low hydrophobicity, the antimicrobial activity can be enhanced by appropriately increasing the number of hydrophobic amino acids, while avoiding the increase of hemolytic activity. Therefore, we replaced the hydrophilic amino acid Asp at position 20 with the hydrophobic amino acid Ile to increase its hydrophobicity, and obtained the rMytichitin-CB-D20I modified peptide (its amino acid sequence is shown in SEQ ID NO.2).

[0028] Based on the rMytichitin-CB-D20I modified peptide, His at position 39 was replaced with Ile to further enhance its hydrophobicity, resulting in the rMytichitin-CB-D20I-H39I modified peptide (its amino acid sequence is shown in SEQ ID NO.4).

[0029] Based on the method of altering hydrophobicity and increasing positive charge, the negatively charged amino acid Asp at position 23 of the rMytichitin-CB-D20I modified peptide was replaced with the positively charged amino acid Arg, resulting in the rMytichitin-CB-D20I-D23R modified peptide (its amino acid sequence is shown in SEQ ID NO.5).

[0030] Example 2: Construction of the modified peptide recombinant expression vector, induction of expression and purification of the modified peptide

[0031] (1) Construction of improved peptide recombinant expression vector

[0032] The designed rMytichitin-CB-D20I, rMytichitin-CB-D23H, rMytichitin-CB-H39I, and rMytichitin-CB-D20I-D23R primers were designed and sent to Anshengda Biotechnology Co., Ltd. (Suzhou, Jiangsu) for synthesis. Using the recombinant pPICZαA-Mytichitin-CB plasmid as a template, the following primers were used: rMytichitin-CB-D20I (FOR: 5'-GGTGCCTTTTATACCATTACCTGCGATAAAAATGTG-3', whose nucleotide sequence is shown in SEQ ID NO.7; REV: 5'-CACATTTTTATCGCAGGTAATGGTATAAAAGGCACC-3', whose nucleotide sequence is shown in SEQ ID NO.11), rMytichitin-CB-D23H primers (FOR: 5'-ACCGATACCTGCCATAAAAATGTGTTTTATC-3', whose nucleotide sequence is shown in SEQ ID NO.8; REV: 5'-GATAAAACACATTTTTATGGCAGGTATCGG T-3', whose nucleotide sequence is shown in SEQ ID NO.12), and rMytichitin-CB-D20I-D23R (FOR: 5'-GGTGC... CTTTTATACCATTACCTGCCGTAAAAATGTGTTTTATCG-3', whose nucleotide sequence is shown in SEQ ID NO.10; REV:5'-CGATAAAACACATTTTTACGGCAGGTAATGGTATAAAAGGCACC-3', whose nucleotide sequence is shown in SEQ ID NO.14) primers were used to perform PCR amplification of the modified peptides, resulting in recombinant expression vectors pPICZαA-rMytichitin-CB-D20I, pPICZαA-rMytichitin-CB-D23H, and pPICZαA-rMytichitin-CB-D20I-D23R;

[0033] Furthermore, using the recombinant pPICZαA-rMytichitin-CB-D20I plasmid as a template, PCR amplification was performed using rMytichitin-CB-H39I primers (FOR: 5'-GAAAAAAATTTGCGGTCGTGGTC-3', whose nucleotide sequence is shown in SEQ ID NO. 9; REV: 5'-GACCACGACCGCAAATTTTTTTC-3', whose nucleotide sequence is shown in SEQ ID NO. 13) to obtain the pPICZαA-rMytichitin-CB-D20I-H39I recombinant expression vector.

[0034] The PCR reaction conditions are as follows:

[0035]

[0036] Electrophoretic verification test of PCR amplification products ( Figure 1 The results showed that the target band appeared at around 4000bp in each modified peptide recombinant expression vector, indicating successful amplification and allowing for the next experimental step.

[0037] After overnight digestion with 2 μL of DpnI, the PCR product was chemically transformed into *E. coli* XL1. Positive clones from LBZ antibiotic plates were picked and inoculated into 5 mL of LB medium containing zeocin. Plasmid mini-extraction was performed, followed by double enzyme digestion (EcoRI / KpnI) for verification. Figure 2 As shown, the target band appeared at 216bp, which initially verified that the plasmid size was correct. The plasmid that was initially verified to be correct was sent for sequencing. The plasmid that was correctly sequenced was linearized by SacI enzyme.

[0038] The linearized plasmid was transferred into Pichia pastoris X-33 via electrochemical conversion. The specific method is as follows: First, a 2mm electroporation cuvette was inverted and placed in a clean bench under UV light and ventilated for drying. 80μL of the prepared competent yeast cells were mixed with 1-2μg of the linearized plasmid, and then added to the electroporation cuvette. The mixture was incubated on ice for 5 minutes. Then, the cuvette was placed in an electroporator for electroporation. The electroporation parameters were: voltage 1500V, resistance 200Ω, capacitance 25μF. After electroporation, 600μL of ice-cold 1M sorbitol was quickly added to the cuvette, and after thorough resuscitation, the mixture was transferred to a 1.5mL EP tube and incubated at 30℃ for 1-2 hours. Spread 200 μL of bacterial culture onto a YPDZ plate (10 g yeast extract, 20 g peptone, 182.1 g sorbitol, and 20 g agar powder were placed in a beaker, and deionized water was added to bring the volume to 900 mL. After stirring well, the mixture was autoclaved at 121°C for 20 min. Once the culture medium cooled to approximately 55°C, 100 mL of sterilized 20% glucose and 100 μg / mL of antibiotic Zeocin were added to each plate in a clean bench, and then dispensed into 90 mm Petri dishes, 25 mL per plate, with three replicates. After incubating upside down at 28°C for 2–3 days, single colonies were picked from the plates for colony PCR to screen for positive clones. The appearance of the target band at 849 bp indicates that the transformant is a positive clone.

[0039] (2) Induction and purification of modified peptides

[0040] The strains expressing the positive modified peptide were selected and cultured in 20 mL of BMGY medium (1% yeast extract, 2% peptone, 1.34% YNB, 0.004% biotin, 1% glycerol, 10 mM potassium phosphate buffer (pH 6.0)) at 28°C on a shaker at 220 rpm for 24 h. A blank control was used. After 24 h, the culture was expanded by transferring 0.5% of the culture to 100 mL of BMGY medium and incubating at 28°C on a shaker at 220 rpm. When the OD600 value reaches 8.0–10.0, the cells are transferred to a sterile 50 mL centrifuge tube and centrifuged at 3500 rpm for 5 min. The BMGY medium is discarded, and the cells are resuspended in 1 mL of BMMY medium (1% yeast extract, 2% peptone, 1.34% YNB, 0.004% biotin, 2% methanol, 10 mM potassium phosphate buffer (pH 6.0)). The cells are then transferred back to BMMY medium and incubated on a shaker at 28℃ and 220 rpm for expression induction. Every 24 h, 100% methanol sterilized by a 0.22 μm filter is added to bring the final concentration to 1.5%. After culturing for 144 h, the supernatant is collected by centrifugation.

[0041] Add 100 mM PMSF to the supernatant to prevent protein degradation, and filter through a 0.22 μm filter membrane in a clean bench. Add 40 mL of yeast supernatant to the prepared column and incubate at room temperature with low-speed shaking for 2–3 h. Wash sequentially with 20, 40, and 60 mM imidazole to remove contaminating proteins, then elute the target protein with elution buffer containing 500 mM imidazole, adding 1 mL of eluent each time, allowing it to stand for 5 min, and collecting the eluent in a centrifuge tube until colorless (G-250 detection). Add the eluent to a 3 kDa ultrafiltration tube and centrifuge at 3500 rpm and 4 °C to concentrate to 1 mL. Replace the eluent by adding 1 mL of replacement buffer (150 mM NaCl, 50 mM PBS, 1 mM DTT, 1 mM EDTA, pH 7.4) and centrifuge at 3500 rpm until imidazole is completely removed.

[0042] Example 3: Determination of Improved Peptide Performance

[0043] (1) Determination of minimum inhibitory concentration

[0044] The test bacteria included Staphylococcus aureus (ATCC 25923), Listeria monocytogenes (ATCC 221633), Bacillus subtilis (151-1), Bacillus subtilis (L300-1), Bacillus subtilis (LZZ133), Escherichia coli (ATCC 10305), Escherichia coli (ATCC 25922), Escherichia coli O157 (ATCC 35150), Cronobacter sakazakii, Cronobacter sakazakii (ATCC 25944), Vibrio parahaemolyticus, Vibrio harveyi (VIB645), Vibrio anguillarum (MCCC.1AO7299), Vibrio anguillarum (LAO7299), Enterococcus faecalis, Salmonella enteritidis (ATCC 13076), Salmonella (ATCC 14028), Pseudomonas fluorescens (CGMCC 1.1802), Pseudomonas tatarsus, and Enterobacter aerogenes.

[0045] According to Table 1 below, 100 μL of the test bacteria diluted 1000-fold (with an absorbance A value of 1.0 at 600 nm) was added to each well of a 96-well plate. Subsequently, 20 μL of Mytinchitin-CB diluted to different concentrations and four rMytinchitin-CB modified peptide samples were added to each well for the experiment, with three replicates for each treatment group. After adding the samples, the 96-well plates were fixed on a shaker and incubated at 37°C for 12 h, and the OD value at 595 nm was measured.

[0046] Table 1. Sample dosage for each treatment

[0047]

[0048]

[0049] After calculating the inhibition rates of different concentrations of the tested bacteria using Equation-1, a graph was plotted with the concentrations of each modified peptide and gentamicin on the x-axis and the inhibition rate on the y-axis (R0). 2 >0.95), calculate the minimum inhibitory concentration of the modified peptide when the inhibition rate is 90%. The results are shown in Table 2.

[0050] As shown in Table 2, all modified peptides exhibit broad-spectrum antibacterial activity, maintaining inhibitory activity against both Gram-positive and Gram-negative bacteria. Specifically, rMytichitin-CB-D20I-D23R shows inhibitory activity against Staphylococcus aureus (ATCC 25923), Listeria monocytogenes (ATCC 221633), Bacillus subtilis (151-1), Bacillus subtilis (L300-1), Bacillus subtilis (LZZ133), Escherichia coli (ATCC 10305), Escherichia coli (ATCC 25922), Escherichia coli O157 (ATCC 35150), Cronobacter sakazakii, Cronobacter sakazakii (ATCC 25944), Vibrio parahaemolyticus, Vibrio harveyi (VIB645), Enterococcus faecalis, and Salmonella enteritidis (ATCC 25923). The antibacterial effects of *Pseudomonas fluorescens* (CGMCC1.1802) and *Pseudomonas torus* were relatively good, with minimum inhibitory concentrations (μg / ml) of 0.35, 0.46, 0.42, 0.35, 0.4, 0.88, 0.51, 0.67, 0.56, 0.35, 0.88, 0.88, 0.51, 0.28, 0.88, and 0.46, respectively. In particular, D20I-D23R showed a significant antibacterial effect against *Staphylococcus aureus* (more than 2-fold increase in activity), with a minimum inhibitory concentration reaching 0.35 μg / ml. Meanwhile, rMytichitin-CB-D23H showed significant antibacterial effects against *Vibrio anguillarum* (MCCC.1AO7299) and *Salmonella*, with minimum inhibitory concentrations of 0.29 μg / ml and 0.17 μg / ml, respectively, significantly lower than the values ​​reported in existing literature.

[0051] Table 2. Minimum Inhibitory Concentration (MIC) of Modified Peptides

[0052]

[0053]

[0054] (2) Determination of minimum bactericidal concentration

[0055] Staphylococcus aureus and Salmonella were selected as the test bacteria. Single colonies of Staphylococcus aureus and Salmonella were picked and cultured in culture medium. When the absorbance at 600 nm reached 1.0, the test bacteria were diluted 1000 times for later use. 50 μL of the test bacterial culture was added to a 1.5 mL EP tube, and 50 μL of different concentrations of antimicrobial peptides (1×MIC, 1.5×MIC, 2×MIC, 2.5×MIC, 3×MIC, 3.5×MIC, 4×MIC, 4.5×MIC, 5×MIC) were added to each tube. The negative control was a replacement buffer (150 mM NaCl, 50 mM PBS, 1 mM DTT, 1 mM EDTA, pH 7.4), and the positive control was 2×MIC (13.2 μg / mL) of gentamicin. Each concentration was repeated in triplicate. After static incubation at 37℃ for 5 hours, 10 μL of the treated bacterial solution was taken and serially diluted from 10 to 1000 times. Then, 100 μL of each solution was spread onto MSA and HE plates, respectively. After static incubation at 37℃ for 12 hours, single colonies on the plates were counted. The lowest protein concentration at which no colonies grew on the plate was the minimum bactericidal concentration.

[0056] The results are shown in Table 3. Regarding the bactericidal activity against Staphylococcus aureus, the minimum bactericidal concentration (MIC) of Mytichitin-CB was 5×MIC. The MICs of the modified peptides rMytichitin-CB-D20I, rMytichitin-CB-D23H, and rMytichitin-CB-D20I-D23R were all increased to 4.5×MIC, 2×MIC, and 2×MIC, respectively. Regarding the bactericidal activity against Salmonella, the MICs of the modified peptides rMytichitin-CB-D20I, rMytichitin-CB-D23H, and rMytichitin-CB-D20I-D23R were all 1×MIC, significantly superior to Mytichitin-CB. Unfortunately, the mutated modified peptide rMytichitin-CB-D20I-H39I only exhibited inhibitory activity and did not have bactericidal activity. The MBC experiment showed that the bactericidal effect of the modified peptides rMytichitin-CB-D20I, rMytichitin-CB-D23H, and rMytichitin-CB-D20I-D23R obtained after molecular modification of Mytichitin-CB was significantly improved, indicating that increasing its hydrophobicity and increasing its charge number can improve its bactericidal ability.

[0057] Table 3. Minimum Bactericidal Concentration (MBC) of Modified Peptides

[0058]

[0059] (3) Sterilization kinetics experiment

[0060] Staphylococcus aureus and Salmonella were selected as the test bacteria. Single colonies of Staphylococcus aureus and Salmonella were picked and cultured in a culture medium. When the absorbance (A value) at 600 nm reached 1.0, the test bacteria were diluted 1000 times for later use. Add 50 μL of bacterial culture to each 1.5 mL EP tube. Add 50 μL of modified peptides (1×MIC, 2×MIC, 5×MIC) at different concentrations to each tube. The negative control is a replacement buffer (150 mM NaCl, 50 mM PBS, 1 mM DTT, 1 mM EDTA, pH 7.4). Gentamicin at 2×MIC (13.2 μg / mL) is used as a positive control. At 0 h, 1 h, 2 h, 3 h, 4 h, and 5 h, take 10 μL of sample, dilute it 10-fold, and spread it on MSA and HE plates. Incubate the plates overnight at 37°C. Count the colonies, organize the experimental data, and construct the bactericidal kinetics diagram as shown below. Figure 3-4 As shown. By Figure 3 It can be seen that the colony count in the negative control increased with the increase of treatment time. In the positive control group treated with 2×MIC gentamicin, Staphylococcus aureus was completely killed within 2 hours, demonstrating rapid action. In contrast, in Figure a, the Mytichitin-CB treatment group showed no bactericidal activity against Staphylococcus aureus at concentrations of 1×MIC and 2×MIC, but at a concentration of 5×MIC, Staphylococcus aureus was completely killed within 5 hours. In Figure b, the modified peptide rMytichitin-CB-D20I showed no bactericidal activity against Staphylococcus aureus at concentrations of 1×MIC and 2×MIC, but at a concentration of 5×MIC, the tested bacteria were completely killed within 4 hours. In Figure c, the modified peptide rMytichitin-CB-D23H killed Staphylococcus aureus within 3 hours at a concentration of 2×MIC, and especially at a concentration of 5×MIC, it killed Staphylococcus aureus within 1 hour, demonstrating rapid action, superior to the positive control group treated with 2×MIC gentamicin. In Figure d, when the modified peptide rMytichitin-CB-D20I-D23R was applied to Staphylococcus aureus at a concentration of 2×MIC for 4 hours, the number of Staphylococcus aureus colonies tended to be 0.

[0061] The effect of Salmonella on Salmonella is determined by Figure 4 The colony count in the negative control group increased over time, while the positive control group treated with 2×MIC gentamicin killed Salmonella at 2 hours. Mytichitin-CB only had bactericidal activity at 5×MIC. Figure 4(a) The modified antimicrobial peptides rMytichitin-CB-D20I, rMytichitin-CB-D23H, and rMytichitin-CB-D20I-D23R exhibit bactericidal effects at a concentration of 1×MIC. Figure 4 (bd). When the treatment group concentration was 5×MIC, the effect on Salmonella was similar to that of 2×MIC gentamicin, and Salmonella could be killed within 2 hours.

[0062] The bactericidal kinetics experiments on Staphylococcus aureus and Salmonella clearly showed that the modified antimicrobial peptides rMytichitin-CB-D20I, rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R had better bactericidal effects than Mytichitin-CB.

[0063] (4) Temperature stability test

[0064] The 1×MIC modified peptide was treated at different temperatures (4℃, 25℃, 37℃, 65℃, 90℃) for 1 h. Gram-positive Staphylococcus aureus (ATCC 25923) and Gram-negative Salmonella (ATCC 14028), with absorbance A values ​​of 1.0 at 600 nm, were diluted 1000-fold and subjected to MIC experiments. The stability at different temperatures was studied by calculating the antibacterial activity. The results are as follows: Figure 5 As shown.

[0065] from Figure 5 As can be seen from Figure a, the modified peptide exhibits an inhibition rate of over 90% against Staphylococcus aureus within the temperature range of 4–65℃. After treatment at 90℃, the inhibition rate of the modified peptide rMytichitin-CB-D20I against Staphylococcus aureus is only about 75%, while the inhibition rates of the modified peptides rMytichitin-CB-D23H, rMytichitin-CB-D20I-H39I, rMytichitin-CB-D20I-D23R, and Mytichitin-CB against Staphylococcus aureus are not affected by temperature. Figure 5 As shown in Figure b, Mytichitin-CB and the modified peptide exhibited inhibition rates of over 90% against Salmonella after treatment at temperatures ranging from 4 to 90°C. This demonstrates that the modified antimicrobial peptide Mytichitin-CB possesses excellent temperature stability, particularly rMytichitin-CB-D23H, rMytichitin-CB-D20I-H39I, and rMytichitin-CB-D20I-D23R, enabling their application in practical production without diminishing activity due to high temperatures.

[0066] (5) pH stability test

[0067] Samples were treated with buffer solutions at pH values ​​of 2, 4, 6, 8, and 10. The antimicrobial peptide was diluted to the minimum inhibitory concentration (MIC). After treatment, the samples were incubated at 37°C for 4 hours, and then the MIC experiment was performed. The stability of the peptide at different pH values ​​was studied by calculating its antimicrobial activity. The results are as follows: Figure 6 As shown.

[0068] pass Figure 6 As shown in Figure a, after treatment with buffer solutions at pH values ​​of 2, 4, and 6, the inhibition rates of Mytichitin-CB and the modified peptides against Staphylococcus aureus were all higher than 98%, indicating that the activity of the modified peptides rMytichitin-CB-D20I, rMytichitin-CB-D23H, rMytichitin-CB-D20I-H39I, and rMytichitin-CB-D20I-D23R was not affected under acidic and neutral conditions. However, when the buffer solution pH was 10, the inhibition rate of Mytichitin-CB, rMytichitin-CB-D20I, and rMytichitin-CB-D20I-H39I against Staphylococcus aureus was lower than 90%, while the modified peptides rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R maintained an inhibition rate higher than 90% even under alkaline conditions, and exhibited good stability in acidic, neutral, and weakly alkaline environments.

[0069] pass Figure 6 As shown in Figure b, Mytichitin-CB and the modified peptides maintained good activity when the pH of the buffer solution was between 2 and 6. At pH 8, the inhibitory effect of rMytichitin-CB-D20I-H39I on Salmonella was less than 30%, indicating that the antibacterial effect of rMytichitin-CB-D20I-H39I against Salmonella (ATCC 14028) was inhibited under weakly alkaline conditions. At pH 10, the inhibition rates of Mytichitin-CB and the modified peptides rMytichitin-CB-D20I, rMytichitin-CB-D20I-H39I, and rMytichitin-CB-D20I-D23R against Salmonella were less than 60%, while rMytichitin-CB-D23H remained unaffected.

[0070] In summary, the inhibitory effects of rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R on Staphylococcus aureus and Salmonella are less affected by pH.

[0071] (6) Protease stability test

[0072] Mytichitin-CB and the modified peptide were dissolved to the MIC concentration using Glycine-HCl (pH=2), PBS (pH=6), PBS (pH=7), and Tris-HCl (pH=8), respectively. Then, 0.126 U each of pepsin (pH=2), papain (pH=6), proteinase K (pH=7), and trypsin (pH=8) were added. After treatment at 37℃ for 2 h, Staphylococcus aureus and Salmonella were used as test strains for MIC experiments. The stability under different enzyme treatments was studied by calculating the antibacterial activity. The results are as follows: Figure 7 As shown. The antibacterial effect of enzyme treatment on Staphylococcus aureus is as follows. Figure 7 As shown in Figure a, the inhibition rates of samples treated with pepsin and papain against Staphylococcus aureus were not affected. However, treatment with proteinase K reduced the inhibition rates of Mytichitin-CB, rMytichitin-CB-D20I, rMytichitin-CB-D23H, and rMytichitin-CB-D20I-H39I against Staphylococcus aureus to varying degrees. Among these, rMytichitin-CB-D20I showed an inhibition rate of only about 60%, failing to achieve a significant antibacterial effect. In addition, trypsin also has a certain impact on the stability of antimicrobial peptides. The inhibition rate of Mytichitin-CB and the modified peptide rMytichitin-CB-D20I-H39I is less than 90%, and the antibacterial effect is significantly reduced. This is mainly because the peptide chains of the antimicrobial peptides Mytichitin-CB and the modified peptide contain peptide bonds composed of carboxyl groups of lysine or arginine. Trypsin can selectively hydrolyze the peptide bonds in the peptide chains, thereby reducing the activity of some antimicrobial peptides. It is worth noting that the activity of the modified peptides rMytichitin-CB-D20I-D23R and rMytichitin-CB-D23H is basically unaffected after protease treatment, indicating that the modified peptides rMytichitin-CB-D20I-D23R and rMytichitin-CB-D23H have good protease stability.

[0073] from Figure 7As shown in Figure b, the modified peptide rMytichitin-CB-D20I-H39I only exhibits certain stability against pepsin treated at pH 2, while its antibacterial activity against Salmonella (ATCC 14028) is easily reduced by weakly acidic, neutral, and weakly alkaline proteases. However, the antibacterial effects of rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R against Salmonella (ATCC 14028) are unaffected after treatment with the four enzymes.

[0074] In summary, the inhibitory effects of rMytichitin-CB-D23H and rMytichitin-CB-D20I-D23R on Staphylococcus aureus and Salmonella were not affected after treatment with different proteases.

[0075] (7) Cytotoxicity test

[0076] African green monkey kidney cells (Vero), bovine kidney cells (MDBK), Coxsbane dog kidney cells (MDCK), and suckling hamster kidney cells (BHK 21) were used as test cells. Cells were cultured in a 37℃ CO2 incubator until they reached the desired number, then seeded into plates. Once the cells reached a density of approximately 50-60%, samples were added. 10 μL of different concentrations of modified peptides (0, 40, 80, 120, 160 μg / mL) were added to each well. PBS solution was used as a negative control, and 1% Triton X-100 was used as a positive control. Each group was in triplicate. After 24 h of cell growth, 10 μL of MTT solution (tetrazolium salt, added in the dark) was added to each well of a 96-well plate, bringing the final concentration to 0.5 μg / mL. After gentle shaking, the plates were incubated in a 37℃ CO2 incubator for 4 h. Protect from light, remove the supernatant, add 100 μL LDMSO to each well, and perform detection using a microplate reader. Shake the plate for 10 min and measure the absorbance at 570 nm. Cell viability is calculated using Equation 2 to assess cytotoxicity. Results are as follows: Figure 8 As shown.

[0077] Cell viability = OD 570 sample / OD 570 PBS × 100% (Equation 2)

[0078] from Figure 8As can be seen, cell survival rate decreased with increasing antimicrobial peptide concentration. rMytichitin-CB-D20I-H39I exhibited the strongest toxicity: at 160 μg / mL, the survival rate of MDBK / BHK-21 / Vero cells was <50%. rMytichitin-CB-D20I and rMytichitin-CB-D20I-D23R showed the best safety profile, with MDCK cell survival rate >80% at 80 μg / mL and MDBK cell survival rate >50% at 160 μg / mL. rMytichitin-CB-D23H showed concentration-dependent toxicity and was most sensitive to Vero cells (survival rate <50% at 160 μg / mL).

[0079] In summary, the low toxicity of the modified peptides rMytichitin-CB-D20I and rMytichitin-CB-D20I-D23R greatly increases the likelihood of their application in clinical research.

Claims

1. The improved Mytichitin-CB antibacterial peptide coding gene is a first gene or a second gene; characterized in that, The nucleotide sequence of the first gene is shown as SEQ ID NO. 15; and the nucleotide sequence of the second gene is shown as SEQ ID NO.

16.

2. The modified Mytichitin-CB antibacterial peptide, which is the first antibacterial peptide or the second antibacterial peptide; characterized in that, The amino acid sequence of the first antibacterial peptide is the amino acid sequence encoded by the first gene of claim 1; and the amino acid sequence of the second antibacterial peptide is the amino acid sequence encoded by the second gene of claim 1.

3. A recombinant plasmid comprising the coding gene of claim 1, being a first recombinant plasmid or a second recombinant plasmid; the first recombinant plasmid comprising the first gene; the second recombinant plasmid comprising the second gene; characterized in that, The expression plasmid of the first recombinant plasmid or the second recombinant plasmid is pPICZ alpha A.

4. A recombinant bacterium comprising the recombinant plasmid of claim 3, which is a first recombinant bacterium or a second recombinant bacterium; the first recombinant bacterium comprises the first recombinant plasmid; the second recombinant bacterium comprises the second recombinant plasmid; characterized in that, The original strain of the recombinant bacteria is Pichia pastoris.

5. The recombinant bacteria of claim 4, wherein, The Pichia pastoris is Pichia pastoris X-33.

6. Use of the coding gene of claim 1, or the antibacterial peptide of claim 2, or the recombinant plasmid of claim 3, or the recombinant bacteria of claim 4 or 5 in the preparation of a preparation for inhibiting bacteria, characterized in that, The bacteria are one or more of Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio anguillarum, Enterococcus faecalis, Pseudomonas tolasii, Enterobacter aerogenes or Salmonella.

7. Use according to claim 6, characterized in that, The bacteria are Staphylococcus aureus or Salmonella.

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