An antibacterial peptide based on uyct1 and a preparation method thereof

Abstract

The present application relates to the technical field of biological medicine, and more particularly to an antibacterial peptide based on UyCT1 and a preparation method thereof.The technical scheme comprises polypeptide UyCT1, polypeptide UyCT1-5K and polypeptide UyCT1-6K, the amino acid sequence of the polypeptide UyCT1 is G-F-W-G-K-L-W-E-G-V-K-N-A-I, the amino acid sequence of the polypeptide UyCT1-5K is G-F-W-K-K-L-W-K-K-V-K-N-A-I, and the amino acid sequence of the polypeptide UyCT1-6K is K-F-W-K-K-L-W-K-K-V-K-N-A-I.The antibacterial peptide is synthesized by Fmoc solid-phase synthesis, and the preparation method is efficient and reliable, simple to operate, has less by-products, and has high yield, the polypeptides UyCT1-5K and UyCT1-6K have broad-spectrum and high-efficiency antibacterial activity on gram-negative bacteria and gram-positive bacteria, the antibacterial activity is significantly improved compared with the parent peptide UyCT1, the cytotoxicity of the polypeptides UyCT1-5K and UyCT1-6K is significantly reduced, the polypeptides have better biocompatibility, provide a new theoretical basis and candidate molecules for the research and development of new antibacterial drugs, and have potential clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an antimicrobial peptide based on UyCT1 and its preparation method. Background Technology

[0002] The problem of multidrug-resistant bacterial infections is becoming increasingly serious due to the improper use of antibiotics currently widely used in clinical practice. Data from the Global Antimicrobial Resistance Surveillance System shows that antibiotic resistance is prevalent in countries around the world. The World Health Organization estimates that by 2050, approximately 70,000 people will die annually from multidrug-resistant bacterial infections, seriously threatening human health and socio-economic development. Antimicrobial peptides, as an important component of the body's natural immune system, have the characteristics of high antimicrobial activity and low resistance rate, and are considered ideal alternatives to antibiotics.

[0003] Scorpion venom peptides are a class of polypeptides derived from scorpion venom. They are widely available and diverse, possessing broad-spectrum antibacterial, anticancer, and anti-inflammatory activities, and hold potential value for drug development. However, existing scorpion venom peptides, such as UyCT1 derived from the venom of the Australian scorpion *Urodacus yaschenkoi*, suffer from high toxicity, poor stability, and difficulty in purification, limiting their clinical application. Therefore, we propose an antimicrobial peptide based on UyCT1 and its preparation method. Summary of the Invention

[0004] The purpose of this invention is to address the problems existing in the background art by proposing an antimicrobial peptide based on UyCT1 and its preparation method.

[0005] The technical solution of this invention: An antimicrobial peptide based on UyCT1, comprising peptide UyCT1, peptide UyCT1-5K, and peptide UyCT1-6K, wherein the amino acid sequence of peptide UyCT1 is GFWGKLWEGVKNAI, the charge number of peptide UyCT1 is +1, and the secondary structure of peptide UyCT1 is an α-helix; the amino acid sequence of peptide UyCT1-5K is GFWKKLWKKVKNAI, the charge number of peptide UyCT1-5K is +5, and the secondary structure of peptide UyCT1-5K is an α-helix; the amino acid sequence of peptide UyCT1-6K is KFWKKLWKKVKNAI, the charge number of peptide UyCT1-6K is +6, and the secondary structure of peptide UyCT1-6K is an α-helix.

[0006] Preferably, the antimicrobial peptide is synthesized using the Fmoc solid-phase synthesis method, comprising the following steps: ① Resin soaking and activation: Place RinkamideMBHAresin resin in the synthesis apparatus and soak it in anhydrous dichloromethane for 4 hours; ② Deprotection of Fmoc protecting group of resin: Remove anhydrous dichloromethane by filtration, add dimethylformamide solution containing 20% ​​piperidine, shake at controlled temperature for 30 min and then filter, and wash the resin with dimethylformamide, isopropanol and anhydrous dichloromethane in sequence. ③ Amino acid activation and coupling: After activating the Fmoc-protected amino acid with HBTU, HOBt and DIEA, it was added to the synthesis instrument and coupled with the resin, and oscillated at 25℃ for 2~12h. ④ Ninhydrin method for detection: Mix the resin with the ninhydrin detection solution and heat at 100℃ for 3 minutes to confirm complete amino acid coupling; ⑤ Repeat steps ② to ④ until all amino acids are linked sequentially; ⑥ Acetylation: Remove the Fmoc protecting group of the last amino acid, add dimethylformamide, dichloromethane, acetic anhydride solution and DIEA, shake at 25°C for 20 min, then wash and dry; ⑦ Antimicrobial peptide shearing: Add resin shearing solution, shake at 25℃ for 4 hours, add pre-cooled anhydrous diethyl ether, centrifuge, and the precipitate is dissolved and freeze-dried to obtain crude antimicrobial peptide; ⑧ Purification: After the crude product is filtered through a 0.22µm filter membrane, it is purified by high performance liquid chromatography. The elution peak with a purity ≥90% is collected and lyophilized. ⑨ Identification: The mass-to-charge ratio of the peptide was determined using a triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system, confirming that the molecular weight error compared to the theoretical value was ≤1 Da.

[0007] Preferably, in step ①, the amount of resin used is 0.25 g (0.2 mmol), and the amount of anhydrous dichloromethane added is 3 mL. In step ③, the amount of Fmoc-protected amino acid used is 0.4 mmol, the amount of HBTU and HOBt added is 0.8 mL each, the amount of DIEA added is 110 μL, and the activation time is 10 min at room temperature. The synthesis instrument is used to realize the automated control of resin activation, deprotection, amino acid coupling, acetylation, and shearing. Its reaction chamber is connected in series with the reagent adding device, temperature control device, shaking device, and vacuum filtration device.

[0008] Preferably, in step ⑦, the resin shearing solution is prepared by mixing trifluoroacetic acid, triisopropylsilane, and purified water in a volume ratio of 90:5:5, with an addition amount of 10 mL. The volume ratio of the pre-cooled anhydrous diethyl ether to the shearing solution is 1:1. The reaction time is 4 h, the centrifugation parameters are 8000 r / min and 4 °C, and the precipitate is dissolved in 50% acetonitrile. The centrifuge equipment is connected to the sample collection device, and the freeze-drying equipment is sealed to the dissolved sample container to achieve vacuum freeze-drying operation.

[0009] Preferably, in step ⑧, the high-performance liquid chromatograph is equipped with a Hypersil 300Å C8 column, mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, mobile phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid, column temperature is 25℃, flow rate is 1mL / min, and detection wavelength is 220nm.

[0010] Preferably, in step 9, the polypeptide dissolution solution is 5% glacial acetic acid, 8% acetonitrile, and 87% purified water, with a sample loading volume of 1µL and a chromatographic flow rate of 0.2mL / min; the sample inlet of the high-performance liquid chromatograph is connected to a filter membrane device, the column outlet is connected in series with a collection device, and the collection device is connected to a lyophilization device; the injection system of the triple quadrupole liquid chromatography-mass spectrometry system is selectively connected to the eluent outlet of the high-performance liquid chromatograph or an independent sample injection device for accurate determination of polypeptide molecular weight.

[0011] Preferably, it also includes secondary structure determination, minimum inhibitory concentration determination, and cytotoxicity detection steps: ⑩ Secondary structure determination: A circular dichroism spectroscopy (CDS) instrument was used. The peptide was dissolved in PBS and 50% trifluoroacetic acid, respectively, to a final concentration of 75 µmol / L. The test wavelength was 190–300 nm, the response time was 1 s, the step size was 0.2 nm, and the scan was performed three times. [θ] was calculated. 222 The relative helicity is analyzed by value analysis. The sample cell of the circular dichroism spectrometer is connected to the spectral detection device and the data acquisition device. The optical path length of the quartz cuvette is 1 cm. ⑪ Determination of minimum inhibitory concentration: Using an ELISA reader in conjunction with a 96-well plate, the final bacterial concentration was adjusted to 1×10⁻⁶. 5 CFU / mL, the working concentration of antimicrobial peptide is 1.5~200µmol / L, and 4 parallel groups are set for each concentration. After incubation at constant temperature and shaking for 24h, the minimum inhibitory concentration is determined by detecting the turbidity of the bacterial solution. ⑫ Cytotoxicity assay: The CCK-8 assay was used. HUVEC cells were seeded in 96-well plates with a final concentration of antimicrobial peptide of 25–125 µmol / L. Each concentration was used in triplicate. After culturing for 24 h, CCK-8 reagent was added and incubated for 2 h. The OD was measured using a microplate reader. 450 nm value and calculate IC 50 The microplate reader is compatible with 96-well plates, and its detection module is connected to the data processing system, which can automatically record absorbance values ​​and perform quantitative analysis.

[0012] Compared with the prior art, the present invention has the following beneficial technical effects: 1. The antimicrobial peptides UyCT1-5K and UyCT1-6K proposed in this invention exhibit broad-spectrum and highly efficient antimicrobial activity against both Gram-negative and Gram-positive bacteria, with significantly enhanced antimicrobial activity compared to the parent peptide UyCT1. Specifically, UyCT1-6K shows an approximately 14.6-fold increase in activity against Gram-negative bacteria and an approximately 16.5-fold increase in activity against Gram-positive bacteria, demonstrating a significant improvement in antimicrobial activity. 2. UyCT1-5K and UyCT1-6K showed significantly reduced cytotoxicity, IC50... 50 The values ​​were 73.44 μmol / L and 85.07 μmol / L, respectively, which were much higher than the 44.79 μmol / L of the parent peptide, indicating better biocompatibility. 3. The antimicrobial peptides were synthesized using the Fmoc solid-phase synthesis method, which is highly efficient and reliable, simple to operate, produces few byproducts, and has a high yield. The purity of the purified peptides is greater than 90%, making it suitable for laboratory preparation and industrial production. 4. This invention provides a new theoretical basis and candidate molecules for the development of novel antibacterial drugs, and has potential clinical application value. Attached Figure Description

[0013] Figure 1 This is a helical diagram of UyCT1 and its derived peptides in this invention; Figure 2 This is a predicted diagram of the secondary structure of UyCT1 and its derived peptides in this invention; Figure 3 This is the chromatographic peak diagram of the UyCT1 peptide in this invention; Figure 4 This is the chromatographic peak diagram of the UyCT1-5K peptide in this invention; Figure 5 This is the chromatographic peak diagram of the UyCT1-6K peptide in this invention; Figure 6 This is a graph showing the mass spectrometry results of UyCT1 in this invention; Figure 7 This is a graph showing the UyCT1-5K mass spectrometry measurement results in this invention; Figure 8 The image shows the mass spectrometry results of UyCT1-6K in this invention. Figure 9 This is a graph showing the results of circular dichroism spectroscopy of the UyCT1 peptide in this invention. Figure 10 This is a circular dichroism chromatogram of the UyCT1-5K peptide in this invention. Figure 11 This is a circular dichroism chromatogram of the UyCT1-6K peptide in this invention. Figure 12 This is a bar graph showing the effect of UyCT1 and its derivative peptides in this invention on the cytotoxicity of HUVEC cells. Detailed Implementation

[0014] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0015] Example like Figure 1-12 As shown, the present invention proposes an antimicrobial peptide based on UyCT1, comprising peptide UyCT1, peptide UyCT1-5K, and peptide UyCT1-6K. The amino acid sequence of peptide UyCT1 is GFWGKLWEGVKNAI, the charge number of peptide UyCT1 is +1, and the secondary structure of peptide UyCT1 is an α-helix; the amino acid sequence of peptide UyCT1-5K is GFWKKLWKKVKNAI, the charge number of peptide UyCT1-5K is +5, and the secondary structure of peptide UyCT1-5K is an α-helix; the amino acid sequence of peptide UyCT1-6K is KFWKKLWKKVKNAI, the charge number of peptide UyCT1-6K is +6, and the secondary structure of peptide UyCT1-6K is an α-helix.

[0016] Based on the design concept, a helical wheel diagram of the UyCT1, UyCT1-5K, and UyCT1-6K peptides was drawn according to their amino acid sequences, as follows: Figure 1 As shown in the diagram, nonpolar amino acids are represented by gray circles, polar amino acids by white hollow circles, and substituted amino acids by triangles. Figure 1 As can be seen, the polar amino acids of the parent peptide and the derived peptide are on one side of the helix, and the nonpolar amino acids are on the opposite side, all of which show good amphiphilicity. The antimicrobial peptide was synthesized using the Fmoc solid-phase method, which included the following steps: ① Resin soaking activation: RinkamideMBHAresin resin was placed in the synthesis instrument and soaked in anhydrous dichloromethane for 4 hours. The amount of resin used in step ① was 0.25 g (0.2 mmol), and the amount of anhydrous dichloromethane added was 3 mL. ② Deprotection of Fmoc protecting group of resin: Remove anhydrous dichloromethane by filtration, add dimethylformamide solution containing 20% ​​piperidine, shake at controlled temperature for 30 min and then filter, and wash the resin with dimethylformamide, isopropanol and anhydrous dichloromethane in sequence. ③ Amino acid activation and coupling: After Fmoc-protected amino acids are mixed and activated with HBTU, HOBt and DIEA, they are added to the synthesis instrument and coupled with the resin. The mixture is kept at 25℃ and shaken for 2~12h. In step ③, the amount of Fmoc-protected amino acids is 0.4mmol, the amount of HBTU and HOBt added is 0.8mL, the amount of DIEA added is 110μL, and the activation time is 10min at room temperature. The synthesis instrument is used to realize the automated control of resin activation, deprotection, amino acid coupling, acetylation and shearing. Its reaction chamber is connected in series with the reagent adding device, temperature control device, shaking device and vacuum filtration device. ④ Ninhydrin method for detection: Mix the resin with the ninhydrin detection solution and heat at 100℃ for 3 minutes to confirm complete amino acid coupling; ⑤ Repeat steps ② to ④ until all amino acids are linked sequentially; The physicochemical properties of UyCT1, UyCT1-5K, and UyCT1-6K peptides were predicted and analyzed using ProtParamtool and HeliQuest websites, as shown in Table 1. These three antimicrobial peptides are all cationic antimicrobial peptides with theoretical isoelectric points between 8.59 and 10.70. They carry 1 to 6 positive charges at pH 7, indicating that they have good solubility under neutral conditions and are not prone to aggregation and precipitation, which is beneficial for the separation, purification, and application of antimicrobial peptides. The hydrophobic moment of a peptide can reflect its amphiphilicity. The hydrophobic moments of UyCT1, UyCT1-5K and UyCT1-6K peptides are 0.694, 0.765 and 0.835, respectively. Compared with the parent peptide, the hydrophobic moments of UyCT1-5K and UyCT1-6K are increased, with UyCT1-6K being higher than UyCT1-5K. This indicates that UyCT1-6K has the greatest amphiphilicity, and theoretically, it is speculated that UyCT1-6K has the strongest membrane-breaking ability. Table 1 Physicochemical properties analysis of UyCT1 and its derived peptides

[0017] ⑥ Acetylation: Remove the Fmoc protecting group of the last amino acid, add dimethylformamide, dichloromethane, acetic anhydride solution and DIEA, shake at 25°C for 20 min, then wash and dry; ⑦ Antimicrobial peptide shearing: Add resin shearing solution, shake at 25℃ for 4 hours, add pre-cooled anhydrous diethyl ether, centrifuge, and the precipitate is dissolved and freeze-dried to obtain crude antimicrobial peptide; In step ⑦, the resin shearing solution is prepared by mixing trifluoroacetic acid, triisopropylsilane and purified water in a volume ratio of 90:5:5, with an addition volume of 10 mL. The volume ratio of pre-cooled anhydrous diethyl ether to the shearing solution is 1:1. The reaction time is 4 h, the centrifugation parameters are 8000 r / min and 4 °C, the precipitate is dissolved in 50% acetonitrile, the centrifuge is connected to the sample collection device, and the freeze-drying device is sealed to the dissolved sample container to achieve vacuum freeze-drying operation. ⑧ Purification: After the crude product is filtered through a 0.22µm filter membrane, it is purified by high performance liquid chromatography. The elution peak with a purity ≥90% is collected and lyophilized. In step ⑧, the high performance liquid chromatograph is equipped with a Hypersil 300Å C8 column, mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, mobile phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid, column temperature is 25℃, flow rate is 1mL / min, and detection wavelength is 220nm. Three peptides were synthesized using the Fmoc solid-phase synthesis method. After shearing and lyophilization, the crude peptide samples were purified by high-performance liquid chromatography. The peak spectra of the three peptides are shown below. Figure 3-5 As shown, the peak elution time of UyCT1 peptide was 17.871 min, with a purity of approximately 97.78%; the peak elution time of UyCT1-5K peptide was 10.061 min, with a purity of approximately 91.78%; and the peak elution time of UyCT1-6K peptide was 13.744 min, with a purity of approximately 90.05%. This indicates that the purity of all three antimicrobial peptides was greater than 90%, indicating good purity. ⑨ Identification: The mass-to-charge ratio of the peptide was determined using a triple quadrupole liquid chromatography-mass spectrometry (LC-MS) system, confirming that the molecular weight error from the theoretical value was ≤1 Da; In step 9, the polypeptide solution is 5% glacial acetic acid, 8% acetonitrile and 87% purified water, the sample loading volume is 1µL, the chromatographic flow rate is 0.2mL / min, the sample injection port of the high performance liquid chromatograph is connected to the filter membrane device, the column outlet is connected in series with the collection device, the collection device is connected to the lyophilization equipment, and the injection system of the triple quadrupole liquid chromatography-mass spectrometry system is selectively connected to the eluent outlet of the high performance liquid chromatograph or the independent sample injection device for the accurate determination of polypeptide molecular weight; Three antimicrobial peptides were analyzed and identified using mass spectrometry. The results are shown below. Figure 6-8In the figure, the vertical axis represents the ion signal intensity, and the horizontal axis represents the ion mass-to-charge ratio (m / z). The relative molecular mass of the target peptide was calculated using the formula, as shown in Table 2. The theoretical molecular weight of UyCT1 peptide is 1645.92 Da, and the mass spectrometry result is 1645.10 Da. The theoretical molecular weight of UyCT1-5K peptide is 1787.22 Da, and the mass spectrometry result is 1787.20 Da. The theoretical molecular weight of UyCT1-6K peptide is 1858.34 Da, and the mass spectrometry result is 1858.10 Da. The difference between the theoretical and measured molecular weights of these three antimicrobial peptides is within 1 Da, which is within the allowable error range, confirming that the peptide synthesis was correct. The formula is: (Where: M represents the molecular weight of the fragmented peptide, Mr represents the molecular weight of the complete peptide, and n represents the charge number) ); Table 2. Mass-to-charge ratio (m / z) and molecular weight (MW) of UyCT1 and its derived peptides

[0018] It also includes secondary structure determination, minimum inhibitory concentration determination, and cytotoxicity testing steps: ⑩ Secondary structure determination: A circular dichroism spectroscopy (CDS) instrument was used. The peptide was dissolved in PBS and 50% trifluoroacetic acid, respectively, to a final concentration of 75 µmol / L. The test wavelength was 190–300 nm, the response time was 1 s, the step size was 0.2 nm, and the scan was performed three times. [θ] was calculated. 222 The relative helicity was analyzed. The sample cell of the circular dichroism spectrometer was connected to the spectral detection device and the data acquisition device. The optical path length of the quartz cuvette was 1 cm. The secondary structures of UyCT1, UyCT1-5K, and UyCT1-6K peptides under simulated hydrophilic and hydrophobic environments were detected using circular dichroism spectroscopy, respectively. Figure 9-11 As shown, using PBS to simulate a hydrophilic environment, the results showed that all three antimicrobial peptides had disordered structures in this environment. Using 50% TFE to simulate a hydrophobic environment, the UyCT1, UyCT1-5K, and UyCT1-6K peptides all exhibited a unique negative absorption peak at 222 nm. Among them, the UyCT1 peptide showed a molar ellipticity of -16625.2 degrees·cm at 222 nm. 2 ·dmol -1 The molar ellipticity of UyCT1-5K is -10385.6 degrees·cm. 2 ·dmol -1 The molar ellipticity of UyCT1-6K is -13430.9 degrees·cm. 2 ·dmol -1 This proves that all three antimicrobial peptides have an α-helix structure; ⑪ Determination of minimum inhibitory concentration: Using an ELISA reader in conjunction with a 96-well plate, the final bacterial concentration was adjusted to 1×10⁻⁶. 5 CFU / mL, the working concentration of antimicrobial peptide is 1.5~200µmol / L, and 4 parallel groups are set for each concentration. After incubation at constant temperature and shaking for 24h, the minimum inhibitory concentration is determined by detecting the turbidity of the bacterial solution. Nine common clinical Gram-negative bacteria (G) were selected. - ) and 6 types of Gram-positive bacteria (G + MIC detection was performed, and the GM values ​​of UyCT1, UyCT1-5K, and UyCT1-6K peptides were calculated. As shown in Tables 3 and 4, the MIC values ​​of UyCT1-5K and UyCT1-6K peptides were significantly lower than those of the parent peptide UyCT1. Compared with UyCT1-5K, UyCT1-6K had even lower MIC values ​​for Escherichia coli, Pseudomonas aeruginosa, Shigella boydii, and Enterococcus faecalis. For 9 types of G - The GM values ​​of UyCT1, UyCT1-5K, and UyCT1-6K peptides were 54.0, 4.7, and 3.7, respectively. UyCT1-5K showed greater resistance to G than UyCT1. - The bacterial activity was increased by approximately 11.5 times, and UyCT1-6K showed greater resistance to Gram-positive bacteria compared to UyCT1. - The bacterial activity increased by approximately 14.6 times for 6 G bacteria. + The GM values ​​of UyCT1, UyCT1-5K, and UyCT1-6K peptides were 44.5, 3.4, and 2.7, respectively. UyCT1-5K showed greater resistance to Gram-positive bacteria than UyCT1. + The bacterial activity was increased by approximately 13.1 times, and UyCT1-6K showed greater resistance to Gram-positive bacteria compared to UyCT1. + The bacterial activity increased by approximately 16.5 times, indicating that these three peptides have an effect on G... - bacteria and G + All bacteria exhibited broad antibacterial activity, with UyCT1-6K peptide showing the strongest antibacterial activity; Table 3 Antibacterial activity of UyCT1 and its derivative peptides against Gram-negative bacteria

[0019] Table 4. Antibacterial activity of UyCT1 and its derivative peptides against Gram-positive bacteria.

[0020] ⑫ Cytotoxicity assay: The CCK-8 assay was used. HUVEC cells were seeded in 96-well plates with a final concentration of antimicrobial peptide of 25–125 µmol / L. Each concentration was used in triplicate. After culturing for 24 h, CCK-8 reagent was added and incubated for 2 h. The OD was measured using a microplate reader. 450 nm value and calculate IC 50The microplate reader is compatible with 96-well plates, and its detection module is connected to the data processing system, which can automatically record absorbance values ​​and perform quantitative analysis. The cytotoxic effects of UyCT1, UyCT1-5K, and UyCT1-6K peptides on HUVEC cells were determined using the CCK-8 assay. Figure 12 As shown, compared with the control group, the cell viability of UyCT1, UyCT1-5K, and UyCT1-6K peptides all showed a decreasing trend, and the IC50 value was significantly lower. 50 The concentrations were 44.79 μmol / L, 73.44 μmol / L, and 85.07 μmol / L, respectively. All three peptides were toxic to HUVEC cells, but the toxicity of UyCT1-5K and UyCT1-6K peptides was significantly lower than that of the parent peptide UyCT1 (P<0.05 or P<0.01). The toxicity of UyCT1-6K peptide was lower than that of UyCT1-5K, which was most significant at 75 μmol / L (P<0.05). The derived peptides UyCT1-5K and UyCT1-6K exhibit broad-spectrum antibacterial activity and a more significant reduction in cytotoxicity, indicating the feasibility of designing and modifying antimicrobial peptides by increasing their positive charge. The amount of positive charge has a significant impact on the bioactivity of antimicrobial peptides. The derived peptide UyCT1-6K demonstrates higher selectivity and has the potential to become a novel antimicrobial peptide. Further in-depth research into the antimicrobial mechanism and targets of derived peptides will provide more reliable experimental evidence for the development of novel antimicrobial drugs.

[0021] In this embodiment, firstly, using the 14-peptide UyCT1 as the parent peptide with the amino acid sequence GFWGKLWEGVKNAI, the electrically neutral glycine G at position 4, the negatively charged glutamic acid E at position 8, and the glycine G at position 9 of UyCT1 were replaced with positively charged lysine K, designed as UyCT1-5K, increasing the charge number from +1 to +5. Subsequently, the glycine G at position 1 of UyCT1-5K was further replaced with lysine K, making the charge number +6, as shown in Table 5. Table 5. Amino acid sequences and charge numbers of UyCT1 and its derivative peptides

[0022] Then, bioinformatics methods were used to quickly and accurately predict the physicochemical properties and structure of antimicrobial peptides, providing a theoretical basis for the design or modification of antimicrobial peptides. The bioinformatics analysis tools used are shown in Table 6. Table 6 Bioinformatics Analysis Tools for Antimicrobial Peptides

[0023] The secondary structures of UyCT1, UyCT1-5K, and UyCT1-6K peptides were predicted and analyzed using the I-TASSER website, such as... Figure 2 As shown, the secondary structures of these three antimicrobial peptides are all α-helices; Next, the UyCT1, UyCT1-5K and UyCT1-6K peptides were synthesized using the 9-fluorenylmethoxycarbonyl protecting group solid-phase synthesis method, with appropriate adjustments made according to experimental needs. Next, the antimicrobial peptide synthesis solution was prepared. The solutions and preparation methods required for the peptide synthesis process are shown in Table 7. Table 7 Preparation of antimicrobial peptide synthesis solution

[0024] In the synthesis of antimicrobial peptides, 0.25 g (0.2 mmol) of Rinkamide MBHAresin resin was first weighed into the synthesis instrument and soaked in 3 mL of anhydrous dichloromethane for 4 h to complete the resin soaking and activation treatment. Then, DCM was removed by vacuum filtration, and 4 mL of DMF shear solution containing 20% ​​piperidine was added. The mixture was then shaken at a controlled temperature for 30 min and the shear solution was removed by vacuum filtration. The resin was washed sequentially with DMF, isopropanol, and DCM to deprotect the Fmoc protecting group. Then, 0.4 mmol of the required amount of Fmoc-AA was weighed and added sequentially with 0.8 mL of HBTU, 0.8 mL of HOBt, and 110 µL of DIEA. The mixture was shaken and incubated at room temperature for 10 min to activate the amino acid. The activated amino acid was then added to a synthesis tube, followed by 3 mL of DCM and 0.5 mL of xylene. The resin was thoroughly soaked and incubated at 25 °C with shaking for 2–12 h. After the reaction was complete, the resin was filtered under reduced pressure and washed to complete the coupling of the amino acid. The resin was then detected using the ninhydrin method. A small amount of resin was placed in an EP tube, and 50 µL of LA, B, and C detection solutions were added, respectively. The mixture was shaken and incubated at 100 °C for 3 min. Observe the resin color. If the resin is bright yellow, the reaction is complete and no free amino groups are exposed, allowing the next amino acid to be linked. If the resin is blue or purple, the reaction is incomplete, with exposed free amino groups present, requiring re-linking of the amino acid. This is achieved by deprotecting the Fmoc protecting group on the resin, repeating the activation, coupling, and detection of amino acids until all amino acids are linked. After the last amino acid is linked, remove the Fmoc protection and proceed with the acetylation reaction. Add 2 mL of DMF and 0.5 mL of DCM to disperse the resin, then add 2 mL of acetic anhydride solution and 800 µL of DIEA. Shake at 25°C for 20 min, wash 3-4 times with DCM, and finally perform ninhydrin detection. If the resin is bright yellow, wash the resin thoroughly with DCM, DMF, DCM, MeOH, and DCM in sequence. Vacuum dry to remove the organic solvent, completing the acetylation process. Add 10 mL of shearing buffer, shake at 25 °C for 4 h, add pre-cooled anhydrous ether and 10 mL of shearing buffer, react for 4 h, let stand for 1 h, and after the ether separates into layers, a precipitate appears. Collect the precipitate, centrifuge (8000 r / min, 4 °C), dissolve in 50% acetonitrile, and freeze-dry under vacuum to obtain crude antimicrobial peptide, thus completing the shearing operation of the antimicrobial peptide. Then dissolve the crude antimicrobial peptide powder in pure water, filter with a 0.22 µm pore size filter membrane to remove insoluble matter, and purify by high performance liquid chromatography and Hypersil C8 column. Mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B is an acetonitrile solution containing 0.1% TFA. The column temperature is 25 °C, the flow rate is 1 mL / min, and the UV absorbance value at 220 nm is detected. After HPLC analysis, the peak area is integrated, the elution peaks are collected and combined, and the peptide with a purity greater than 90% is freeze-dried and identified by mass spectrometry. Mass spectrometry was used to calculate the molecular mass of the peptide by determining the ion mass-to-charge ratio. The peptide powder was identified by mass spectrometry using a Shimadzu LCMS-2020 triple quadrupole liquid chromatography-mass spectrometry system. The peptide powder was dissolved in 5% glacial acetic acid + 8% acetonitrile + 87% purified water, with a sample loading volume of 1µL, a temperature of 25℃, and a chromatographic flow rate of 0.2mL / min. The secondary structure of the antimicrobial peptide was then determined using a circular dichroism spectroscopy (CDS) instrument. The peptide samples were accurately weighed and dissolved in PBS (simulated as a hydrophilic environment) and 50% trifluoroethanol (simulated as a hydrophobic environment), respectively. The final concentration of the peptide samples was 75 µmol / L. The wavelength range was 190–300 nm, the response time was 1 s, the step size was 0.2 nm, and the scans were performed three times. [θ] was calculated according to the formula. 222 The value of can be used to analyze the relative helicity of UyCT1 and its derived peptides, as shown in the formula:

[0025] (In the formula: [θ]) 222 This represents the average molar ellipticity of residues at a wavelength of 222 nm. The ellipticity is represented by l, the optical path length of the quartz cuvette is represented by c. M (This indicates the concentration of the polypeptide sample, in mol / L; n represents the number of amino acid residues in the polypeptide). The minimum inhibitory concentration (MIC) of antimicrobial peptides was determined using the microbroth dilution method recommended by the American Committee for Clinical Laboratory Standards (CCLSI). The method was modified as needed for the experiment. Single colonies were picked and added to MH liquid medium, incubated at a constant temperature and shaken until the logarithmic growth phase. The bacterial OD value was measured, and the final bacterial concentration was adjusted to 1 × 10⁻⁶ based on the concentration standard curve for this type of bacteria. 5 CFU / mL, the antimicrobial peptide was dissolved in a 0.4% BSA solution containing 0.02% acetic acid, with working concentrations of 200, 100, 50, 25, 12.5, 6.5, 3.5, and 1.5 µmol / L. 90 μL of the diluted bacterial solution was added to each well of a 96-well plate, followed by 10 μL of the antimicrobial peptide. Four parallel groups were set up for each concentration as the experimental group. The negative control group consisted of 100 μL of bacterial solution. After plating, the plates were incubated at a constant temperature with shaking for 24 h. The turbidity of the bacterial solution in each well was observed. The lowest concentration with clear and transparent bacterial solution was taken as the MIC of the antimicrobial peptide. The geometric mean (GM) of the three antimicrobial peptide MICs was calculated using the formula:

[0026] (Where: GM represents the geometric mean; x1~xn represent the MIC values ​​of the antimicrobial peptide against different bacteria;) (Indicates the number); The CCK-8 assay was used to detect the toxic effects of UyCT1 and its derivative peptides on normal cells. HUVECs were selected as test cells and cultured in DMEM-H medium containing 10% fetal bovine serum. The cells were seeded into 96-well plates and allowed to adhere fully. The antimicrobial peptides were dissolved in FBS-free medium to final concentrations of 125, 100, 75, 50, and 25 µmol / L, and added to the 96-well plates. A 0 µmol / L concentration was used as the control group. Each concentration was used in triplicate. After 24 h of culture, the peptide-containing medium was discarded, and 100 μL of medium and 10 μL of CCK-8 reagent were added to each well. The cells were incubated for 2 h, and the OD value was measured using a microplate reader. 450 nm value, calculate IC 50 value; Finally, all experiments were repeated three times. Data were analyzed using Origin 2022 and GraphPad Prism 8.0 software. Experimental results are expressed as mean ± standard deviation. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 indicated that the difference was statistically significant.

[0027] The above specific embodiments are merely preferred embodiments of the present invention. Based on the technical solutions of the present invention and the relevant teachings of the above embodiments, those skilled in the art can make various alternative improvements and combinations to the above specific embodiments.

Claims

1. An antimicrobial peptide based on UyCT1, characterized in that: The formulation includes peptides UyCT1, UyCT1-5K, and UyCT1-6K. The amino acid sequence of peptide UyCT1 is GFWGKLWEGVKNAI, the charge number of peptide UyCT1 is +1, and the secondary structure of peptide UyCT1 is an α-helix. The amino acid sequence of peptide UyCT1-5K is GFWKKLWKKVKNAI, the charge number of peptide UyCT1-5K is +5, and the secondary structure of peptide UyCT1-5K is an α-helix. The amino acid sequence of peptide UyCT1-6K is KFWKKLWKKVKNAI, the charge number of peptide UyCT1-6K is +6, and the secondary structure of peptide UyCT1-6K is an α-helix.

2. The method for preparing antimicrobial peptides according to claim 1, characterized in that, The antimicrobial peptide is synthesized using the Fmoc solid-phase synthesis method, including the following steps: ① Resin soaking and activation: Place RinkamideMBHAresin resin in the synthesis apparatus and soak it in anhydrous dichloromethane for 4 hours; ② Deprotection of Fmoc protecting group of resin: Remove anhydrous dichloromethane by filtration, add dimethylformamide solution containing 20% ​​piperidine, shake at controlled temperature for 30 min and then filter, and wash the resin with dimethylformamide, isopropanol and anhydrous dichloromethane in sequence. ③ Amino acid activation and coupling: After activating the Fmoc-protected amino acid with HBTU, HOBt and DIEA, it was added to the synthesis instrument and coupled with the resin, and oscillated at 25℃ for 2~12h. ④ Ninhydrin method for detection: Mix the resin with the ninhydrin detection solution and heat at 100℃ for 3 minutes to confirm complete amino acid coupling; ⑤ Repeat steps ② to ④ until all amino acids are linked sequentially; ⑥ Acetylation: Remove the Fmoc protecting group of the last amino acid, add dimethylformamide, dichloromethane, acetic anhydride solution and DIEA, shake at 25°C for 20 min, then wash and dry; ⑦ Antimicrobial peptide shearing: Add resin shearing solution, shake at 25℃ for 4 hours, add pre-cooled anhydrous diethyl ether, centrifuge, and the precipitate is dissolved and freeze-dried to obtain crude antimicrobial peptide; ⑧ Purification: After the crude product is filtered through a 0.22µm filter membrane, it is purified by high performance liquid chromatography. The elution peak with a purity ≥90% is collected and lyophilized. ⑨ Identification: The mass-to-charge ratio of the peptide was determined using a triple quadrupole liquid chromatography-mass spectrometry (LC-MS) instrument, confirming that the molecular weight error from the theoretical value was ≤1 Da.

3. The antimicrobial peptide based on UyCT1 and its preparation method according to claim 2, characterized in that, In step ①, the amount of resin used is 0.25 g (0.2 mmol), and the amount of anhydrous dichloromethane added is 3 mL. In step ③, the amount of Fmoc-protected amino acid used is 0.4 mmol, the amount of HBTU and HOBt added is 0.8 mL each, the amount of DIEA added is 110 μL, and the activation time is 10 min at room temperature. The synthesis instrument is used to realize the automated control of resin activation, deprotection, amino acid coupling, acetylation, and shearing. Its reaction chamber is connected in series with the reagent adding device, temperature control device, shaking device, and vacuum filtration device.

4. The antimicrobial peptide based on UyCT1 and its preparation method according to claim 2, characterized in that, In step ⑦, the resin shearing solution is prepared by mixing trifluoroacetic acid, triisopropylsilane, and purified water in a volume ratio of 90:5:5, with an addition volume of 10 mL. The volume ratio of the pre-cooled anhydrous diethyl ether to the shearing solution is 1:

1. The reaction time is 4 h, the centrifugation parameters are 8000 r / min and 4 °C, and the precipitate is dissolved in 50% acetonitrile. The centrifuge equipment is connected to the sample collection device, and the freeze-drying equipment is sealed to the dissolved sample container to achieve vacuum freeze-drying operation.

5. The antimicrobial peptide based on UyCT1 and its preparation method according to claim 2, characterized in that, In step ⑧, the high-performance liquid chromatograph is equipped with a Hypersil 300Å C8 column, mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, mobile phase B is an acetonitrile solution containing 0.1% trifluoroacetic acid, column temperature is 25℃, flow rate is 1mL / min, and detection wavelength is 220nm.

6. The antimicrobial peptide based on UyCT1 and its preparation method according to claim 2, characterized in that, In step 9, the polypeptide solution is composed of 5% glacial acetic acid, 8% acetonitrile, and 87% purified water, with a sample loading volume of 1 µL and a chromatographic flow rate of 0.2 mL / min. The sample inlet of the high-performance liquid chromatograph is connected to a filter membrane device, the column outlet is connected in series with a collection device, and the collection device is connected to a lyophilization device. The injection system of the triple quadrupole liquid chromatography-mass spectrometry system is selectively connected to the eluent outlet of the high-performance liquid chromatograph or an independent sample injection device for the accurate determination of polypeptide molecular weight.

7. The preparation method according to claim 2, characterized in that, It also includes secondary structure determination, minimum inhibitory concentration determination, and cytotoxicity testing steps: ⑩ Secondary structure determination: A circular dichroism spectroscopy (CDS) instrument was used. The peptide was dissolved in PBS and 50% trifluoroacetic acid, respectively, to a final concentration of 75 µmol / L. The test wavelength was 190–300 nm, the response time was 1 s, the step size was 0.2 nm, and the scan was performed three times. [θ] was calculated. 222 The relative helicity is analyzed by value analysis. The sample cell of the circular dichroism spectrometer is connected to the spectral detection device and the data acquisition device. The optical path length of the quartz cuvette is 1 cm. ⑪ Determination of minimum inhibitory concentration: Using an ELISA reader in conjunction with a 96-well plate, the final bacterial concentration was adjusted to 1×10⁻⁶. 5 CFU / mL, the working concentration of antimicrobial peptide is 1.5~200µmol / L, and 4 parallel groups are set for each concentration. After incubation at constant temperature and shaking for 24h, the minimum inhibitory concentration is determined by detecting the turbidity of the bacterial solution. ⑫ Cytotoxicity assay: The CCK-8 assay was used. HUVEC cells were seeded in 96-well plates with a final concentration of antimicrobial peptide of 25–125 µmol / L. Each concentration was used in triplicate. After culturing for 24 h, CCK-8 reagent was added and incubated for 2 h. The OD was measured using a microplate reader. 450 nm value and calculate IC 50 The microplate reader is compatible with 96-well plates, and its detection module is connected to the data processing system, which can automatically record absorbance values ​​and perform quantitative analysis.

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