A group of low-toxic broad-spectrum antibacterial peptides with proline, glycine and palindromic sequence and application thereof

Abstract

The application discloses a group of low-toxicity broad-spectrum antibacterial peptides with proline, glycine and palindrome sequences and application thereof. A group of low-toxicity broad-spectrum antibacterial peptides with proline, glycine and palindrome sequences are obtained by inserting 2-3 Pros in the center and symmetric positions of the sequence G(LK) n (KL) n G-NH2, and the group is marked as GnPm; wherein n is 5 or 6, and the G at the two ends of the sequence can be removed; the antibacterial peptides are marked as G4P1, G5P2, 5P2, G6P2 and G6P3. In-vitro antibacterial experiments show that the antibacterial peptides G4P1, G5P2, 5P2, G6P2 and G6P3 all have broad-spectrum antibacterial activity, low hemolytic toxicity, low cytotoxicity, high serum stability and low drug resistance occurrence. In-vivo acute toxicity experiment results show that, compared with a control drug Polymyxin B, the antibacterial peptides have higher in-vivo safety. Therefore, the antibacterial peptides have a good application prospect in preparation of clinical antibacterial drugs, and are expected to become new antibiotic candidate drugs.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and relates to a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences. This invention also relates to the application of these antimicrobial peptides in the preparation of clinical antimicrobial drugs. Background Technology

[0002] Antibiotics are key drugs for treating bacterial infections, but their overuse and limited mechanisms of action have led to a large number of drug-resistant bacteria, posing a major global health crisis. More than 700,000 people die annually worldwide from drug-resistant bacterial infections, and if left unchecked, the death toll could reach 10 million by 2050 (Antibiotics, 2022, 12, 1-30). Antimicrobial peptides (AMPs) are a class of small molecule polypeptides widely present in the innate immune system of organisms. With their unique advantages such as broad-spectrum antibacterial activity, rapid bactericidal action, and low likelihood of inducing resistance, they have become important candidate drugs for addressing the global antibiotic resistance crisis. Unlike traditional antibiotics, antimicrobial peptides, with their drastically different modes of action, have become a focus of research and development for novel antimicrobial drugs. However, natural antimicrobial peptides have disadvantages such as poor stability and low biocompatibility. Therefore, research on antimicrobial peptides has gradually shifted from natural extraction to artificial design, structural modification, and targeted engineering. A series of optimized molecules, such as cyclic lipopeptides, hinge peptides, and short-chain antimicrobial peptides, have emerged, and some candidate molecules have entered the preclinical and clinical research stages, showing good translational prospects (BiotechnologyAdvances, 2022, 59:107968.).

[0003] Existing research has confirmed that the secondary structure of peptide chains plays a crucial role in their interaction with bacterial cell membranes. This secondary structure directly affects the recognition, binding, and disruption efficacy of antimicrobial peptides against bacterial cell membranes, thus influencing their antimicrobial activity, stability, and biocompatibility. Currently, numerous studies have focused on sequence design for single conformations of antimicrobial peptides. For example, the Motoharu team introduced non-natural amino acids to form carbon-carbon crosslinks, fixing the helical conformation of antimicrobial peptides to enhance their antimicrobial properties and membrane selectivity (Nature Biotechnology). 37(10), 1186-1197; Macyszyn et al. stabilized the membrane-active secondary structure of cell-penetrating peptide (KFF)3K by introducing hydrocarbon staples, and found that it was resistant to proteases (Scientific Reports, 2023, 13. 14826); Other studies have focused on fixing β-sheet, α-helix conformations or designing self-assembled peptides, attempting to improve the performance of antimicrobial peptides by strengthening a single conformation (Scientific Reports, 2024, 14, 9701; Science Advances, 2023, 9, e8782). However, this design approach based on a single conformation generally lacks fine-grained control over the secondary structure of peptide chains. It cannot precisely regulate the dynamic changes in peptide chain conformation according to the bacterial cell membrane environment and functional requirements. This results in gaps in the study of the structure-activity relationship between peptide secondary structure and antibacterial activity and toxicity, making it difficult to clarify the specific impact of different conformational parameters on peptide chain function. Consequently, it is impossible to achieve a precise balance between antimicrobial peptide activity, stability, and toxicity, which is detrimental to the performance optimization and clinical translation of antimicrobial peptides. Therefore, fine-grained control of the secondary structure of antimicrobial peptides and in-depth exploration of their structure-activity relationship have become key issues urgently needing to be addressed in the field of antimicrobial peptide design, and have significant practical implications for the rational design of high-performance antimicrobial peptides. Summary of the Invention

[0004] The purpose of this invention is to provide a group of low-toxicity, broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences.

[0005] Another object of the present invention is to provide the application of the above-mentioned antimicrobial peptides in the preparation of clinical antimicrobial drugs.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: I. Design of a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences This invention provides a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences, whose general sequence formula is: G(LK). n (KL) n G-NH2 (n=2, 3, 4, 5, 6), where the Gs at both ends of the sequence can be removed, 0 to 3 Pros are inserted at the center and symmetrical positions of the sequence, and the peptide conformation, charge and hydrophobicity are optimized by regulating the number of P and LK, and 20 antimicrobial peptides are synthesized and labeled as GnPm (n represents the number of LK repeats and m represents the number of Pros).

[0007] Preferably, the present invention provides a group of low-toxicity, broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences, which are expressed through the sequence G(LK). n (KL) nThe G-NH2 sequence is obtained by inserting 2-3 Pros at the center and symmetrical positions, and is labeled as GnPm; where n is 5 or 6, and the Gs at both ends of the sequence can be removed.

[0008] Preferably, the present invention provides a group of low-toxicity broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences, the amino acid sequences of which are as follows: Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Lys-Leu-Lys -Leu-Lys-Leu-Lys-Leu-Gly-CONH2 (SEQ ID NO. 1), labeled G4P1; Or: Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Gly-CONH2 (SEQ ID NO.2), labeled G5P2; Or: Leu-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Leu-CONH2 (SEQ ID NO.3), marked as 5P2; Or: Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Gly-CONH2 (SEQ ID NO.4), labeled G6P2; Or: Gly-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Lys-Leu-Lys-Leu -Lys-Leu-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Gly-CONH2 (SEQ ID NO. 5), labeled G6P3.

[0009] II. Preparation of a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences This invention discloses a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences, synthesized using Fmoc-Rink-MBHA Resin as a raw material and employing the classic Fmoc solid-phase peptide synthesis method. During peptide synthesis, HOBt / HBTU was used as a condensing agent for amino acid coupling, secondary amines were identified using the ninhydrin colorimetric method, and peptides were sequentially condensed according to their sequences to obtain peptides linked to MBHA resin. The peptides were then cleaved from the MBHA resin and purified by HPLC.

[0010] III. Application of a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences 1. In vitro antibacterial test To evaluate the in vitro antimicrobial activity of the antimicrobial peptide, the minimum inhibitory concentration (MIC) of the antimicrobial peptide against the standard strain was determined using the classic micro-dilution method. Polymyxin B was used as a positive control. The experiment was repeated three times in parallel, and the results are shown in Table 1.

[0011] Table 1. Minimum inhibitory concentrations (MICs) of the antimicrobial peptides of this invention against common standard strains. μ M) Table 1 shows that the antimicrobial peptides of the present invention have strong inhibitory effects on both Gram-positive and Gram-negative bacteria, exhibiting broad-spectrum antimicrobial activity.

[0012] 2. In vitro toxicity test (1) Hemolytic activity test To investigate the toxicity of the antimicrobial peptide synthesized in this invention to normal mammalian cells, the hemolysis of the antimicrobial peptide was tested after co-incubation with a PBS suspension of 8% mouse erythrocytes for 1 h. The results are shown below. Figure 6 .

[0013] Figure 6 The results showed that only G6P2 (the concentration of HC that causes 10% hemolysis of red blood cells) was present. 10 =71.45 μ M) exhibited some hemolytic toxicity, while the remaining peptide chains showed no significant hemolysis even at a concentration of 256 μM, indicating good safety. The hemolytic activity results demonstrate that the antimicrobial peptides of this invention have good safety.

[0014] (2) Cytotoxicity test To investigate the toxicity of the antimicrobial peptide of this invention to normal human cells, the cytotoxicity of the antimicrobial peptide of this invention to HK-2 cells was tested, and the results are shown below. Figure 7 .

[0015] Figure 7 The results indicate that G6P2 (IC) 50The value is 128 μ M), exhibiting some cytotoxicity, while the remaining sequences, even at 128, showed some cytotoxicity. μ Even at the concentration of M, the cell survival rate remains above 50%.

[0016] 3. Serum stability test To investigate the stability of the preferred antimicrobial peptides G5P2 and 5P2 (which exhibit the best in vitro antimicrobial activity and low hemolytic and cytotoxic toxicity) in serum, the stability of G5P2 and 5P2 with mouse serum was tested after co-incubation at 37°C for 1 h and 6 h, and the peptide pair was then measured. S. aureus and E. coli The changes in the antibacterial ability are shown in Table 2.

[0017] Table 2. Minimum inhibitory concentrations of the antimicrobial peptides G5P2 and 5P2 of the present invention after serum treatment. Table 2 shows that the MIC values ​​of G5P2 and 5P2 against Staphylococcus aureus doubled after 1 h and 6 h of incubation with serum, while the MIC values ​​against Escherichia coli remained unchanged after 1 h of incubation with serum and doubled after 6 h, with relatively small changes. The experimental results indicate that G5P2 and 5P2 have a certain degree of stability in serum and are not easily degraded or inactivated by enzymes in serum.

[0018] 4. Induction of drug resistance experiment Due to their unique membrane-breaking mechanism, antimicrobial peptides are difficult for bacteria to develop resistance to. To investigate the development of resistance to the antimicrobial peptides of this invention under continuous use, this invention compared G5P2 and 5P2 with… S. aureus ATCC 25923 and E. coli The changes in the minimum inhibitory concentration (MIC) of ATCC 25922 over 15 days of continuous treatment were determined and recorded. The determination method used antibiotics Rifampicin, Gentamicin, and Polymyxin B as controls. The results are shown below. Figure 8 .

[0019] Figure 8 The results showed that when antibiotics were administered alone, rifampin had an effect on... S. aureus The MIC of gentamicin increased to over 4000-fold on day 13 and continued to rise during subsequent passages. E. coli The MIC of the two strains increased 16-fold in 6 days, indicating the development of resistance; while the MIC values ​​of the antimicrobial peptides G5P2 and 5P2 did not change significantly for the two strains, and no resistance was developed. Figure 9 The results showed that the antimicrobial peptide of the present invention, when used in combination with traditional antibiotics, effectively inhibited the growth of bacteria. S. aureus and E. coliThe MIC values ​​did not change significantly, maintaining good activity, indicating that the combined use of antimicrobial peptides can effectively inhibit the development of bacterial resistance to traditional antibiotics. Therefore, the antimicrobial peptides G5P2 and 5P2 of this invention are not prone to inducing bacterial resistance and have an inhibitory effect on the induction of resistance to traditional antibiotics.

[0020] 5. Acute toxicity test in mice The experimental mice were male BALB / c mice, weighing 18-22g, and were fed in accordance with the Lanzhou University Laboratory Animal Ethics Management Regulations.

[0021] Acute toxicity pre-test in mice: Three mice per group were given a single intraperitoneal injection of antimicrobial peptides G5P2 and 5P2 solutions prepared with physiological saline. Mortality was observed after 7 days. Polymyxin B was used as the control drug. The maximum dose that caused all mice to die and the minimum dose that did not cause all mice to die were determined, and the upper and lower limits of the dose were determined.

[0022] Formal acute toxicity test in mice: The LD50 of G5P2 was obtained based on preliminary experimental results. 50 The dosage range is 71.7-109.35 mg / kg, and the LD50 of 5P2 is... 50 The dosage range is 98.42-121.5 mg / kg, and the LD50 of the control drug Polymyxin B is... 50 The dosage range was 15-30 mg / kg. Based on the dosage range of G5P2, the common ratio for grouping was calculated to be 1.2435, dividing the mice into three groups with doses ranging from low to high: 71.71 mg / kg, 87.93 mg / kg, and 109.35 mg / kg. The common ratio for 5P2 was 1.11, dividing the mice into three groups with doses ranging from low to high: 98.42 mg / kg, 109.35 mg / kg, and 121.5 mg / kg. The common ratio for the control drug Polymyxin B was 1.2667, dividing the mice into four groups with doses ranging from low to high: 15 mg / kg, 19 mg / kg, 24 mg / kg, and 30 mg / kg. Ten mice were administered each dose of each drug to each group via intraperitoneal injection. Mice in each group were observed for mortality within 14 days, and the LD50 was recorded and calculated. 50 The results are shown below. Figure 10 .

[0023] Figure 10The results showed that in the G5P2 group, mice were injected with doses of 70.71, 87.93, and 109.35 mg / kg, respectively. At a dose of 70.71 mg / kg, 90% of the mice survived, but at a dose of 109.35 mg / kg, 90% of the mice died within three days. In the 5P2 group, mice had an 80% survival rate at a dose of 98.42 mg / kg, but all died within six days after injection at a dose of 121.5 mg / kg; at a dose of 109.35 mg / kg, the survival rate was 20%. Conversely, mice in the polymyxin B group had a 90% survival rate at a dose of 15 mg / kg, a 40% survival rate at 19 mg / kg, a 10% mortality rate at 24 mg / kg, and a 0% mortality rate at 30 mg / kg. The calculated LD50 values ​​for the antimicrobial peptides G5P2 and 5P2, and the control drug Polymyxin B in BALB / c mice were determined to be... 50 The LD values ​​were determined to be 89.868, 116.485, and 18.478 mg / kg, respectively, indicating the LD50 of the antimicrobial peptide G5P2 of this invention. 50 The LD50 value of the antimicrobial peptide 5P2 of this invention is approximately 4.86 times that of the control drug Polymyxin B. 50 The value was approximately 6.3 times that of the control drug Polymyxin B, indicating that the antimicrobial peptide of the present invention has higher safety in mice compared with the control drug Polymyxin B.

[0024] In summary, the unique rigid turn structure of proline has a significant impact on the bioactivity of peptides. Introducing proline into antimicrobial peptides (AMPs) can effectively regulate their structure and thus affect their activity. This invention employs a de novo design approach, systematically designing 20 antimicrobial peptides with palindromic sequences containing proline and glycine. The aim is to systematically study the regulatory effects of adjusting sequence length and the introduction of proline and glycine on the secondary structure, hydrophobicity, charge, and stability of antimicrobial peptides, thereby investigating the influence of these factors on the antimicrobial properties of the peptides. Preferably, this invention uses sequence G(LK)... n (KL) nInserting 2-3 Pro molecules at the central and symmetrical positions of G-NH2 yields a group of low-toxicity, broad-spectrum antimicrobial peptides with proline, glycine, and palindromic sequences, labeled GnPm; where n is 5 or 6, and the G molecules at both ends of the sequence can be removed; these antimicrobial peptides are labeled G4P1, G5P2, 5P2, G6P2, and G6P3. In vitro antibacterial experiments show that antimicrobial peptides G4P1, G5P2, 5P2, G6P2, and G6P3 all possess broad-spectrum antimicrobial activity. The preferred representative peptide G5P2 and its control peptide 5P2 (without G at the peptide chain terminal) exhibit low hemolytic toxicity, low cytotoxicity, high serum stability, and low incidence of drug resistance. In vivo acute toxicity experiments show that the antimicrobial peptides G5P2 and 5P2 of this invention have higher in vivo safety compared to the control drug Polymyxin B. Therefore, they have excellent application prospects in the preparation of clinical antimicrobial drugs and are expected to become novel antibiotic candidates. Attached Figure Description

[0025] Figure 1 This is the mass spectrum of the antimicrobial peptide G4P1 of this invention; Figure 2 This is the mass spectrum of the antimicrobial peptide G5P2 of this invention; Figure 3 This is the mass spectrum of the antimicrobial peptide 5P2 of the present invention; Figure 4 This is the mass spectrum of the antimicrobial peptide G6P2 of the present invention; Figure 5 This is the mass spectrum of the antimicrobial peptide G6P3 of this invention; Figure 6 The results show the hemolytic activity of the antimicrobial peptide of this invention on mouse erythrocytes after incubation for 1 hour. Figure 7 These are the results of the cytotoxicity experiment of the antimicrobial peptide of this invention on HK-2 cells; Figure 8 This is the result of resistance induced by the antimicrobial peptides G5P2 and 5P2 of this invention, and by traditional antibiotics alone; Figure 9 This is the result of inducing drug resistance by combining the antimicrobial peptides G5P2 and 5P2 of this invention with traditional antibiotics; Figure 10 The figure shows the results of an in vivo acute toxicity experiment of the antimicrobial peptides G5P2, 5P2 and Polymyxin B of the present invention on BALB / c mice. Detailed Implementation

[0026] The synthesis of the low-toxicity broad-spectrum antimicrobial peptide with proline, glycine, and palindromic sequences of the present invention will be further illustrated below through specific embodiments.

[0027] Example 1: Synthesis of antimicrobial peptide G4P1 (1) Activation of resin.

[0028] Weigh 0.42 g of MBHA resin (0.48 mmol / g), place it at the bottom of the solid phase synthesizer, add DCM to swell for 30 min, dry under vacuum, wash with DMF and dry under vacuum, repeat 3 times, and identify the resin by ninhydrin colorimetric method. If the resin is colorless, it indicates that it can be used normally.

[0029] (2) Synthesis of Fmoc-G4P1-MBHA The swollen resin was deprotected three times with a DMF solution containing 20% ​​piperidine, dried, and washed three times with DMF. The resin was then identified using the ninhydrin colorimetric method; a colored resin indicated that the Fmoc protecting group had been removed. Three times the excess of Gly, HOBt, and HBTU were completely dissolved in DMF, and six times the excess of DIEA was added for full activation. The mixture was immediately added to the synthesizer, and the reaction was carried out at room temperature with stirring for 1 hour under argon protection. After the reaction, the resin was identified using the ninhydrin colorimetric method; a colorless and transparent resin indicated successful Gly condensation, yielding Fmoc-Gly-MBHA. Following the above method, Gly, Leu, Lys, Leu, Lys, Leu, Lys, Leu, Leu, Pro, Lys, Leu, Lys, Leu, Leu, Leu, Leu, Leu, Gly are condensed sequentially to obtain Fmoc-Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Gly-MBHA.

[0030] (3) Peptide cleavage The obtained Fmoc-Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Gly-MBHA was deprotected three times with a DMF solution containing 20% ​​piperidine. After drying, the resin was washed three times with DMF and dried again. The resin was identified using the ninhydrin colorimetric method; a blue-purple color indicated that the Fmoc protecting group had been removed. The resin was washed three times alternately with DCM and methanol and then vacuum-dried for 1 hour. After the resin was dried into dry granules, 10 mL of cleavage reagent (TFA:Tris:water) = 9.5:0.25:0.25 (v:v:v) was added, and the mixture was reacted for 3 hours with slow stirring every 20 minutes. After the reaction time was reached, the cleavage reagent was collected, extracted with diethyl ether, and then freeze-dried to obtain crude peptide powder.

[0031] (4) Peptide purification The crude peptide was purified by RP-HPLC. The RP-HPLC purification conditions were: mobile phase A: 1‰ TFA / acetonitrile, mobile phase B: 1‰ TFA / water, using linear gradient elution. The eluent from the target peak was collected, lyophilized, and G4P1 was obtained. Its mass spectrum is shown below. Figure 1 As shown, the theoretical molecular weight calculation of G4P1 is 2159, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0032] Example 2: Synthesis of antimicrobial peptide G5P2 (1) The activation of the resin is the same as in Example 1.

[0033] (2) The synthesis of Fmoc-G5P2-MBHA was carried out by sequentially condensing Gly, Leu, Lys, Leu, Lys, Leu, Lys, Leu, Pro, Lys, Leu, Lys, Leu, Leu, Lys, Pro, Leu, Lys, Leu, Leu, Leu, Lys, Leu, Gly to obtain Fmoc-Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Gly-MBHA.

[0034] (3) The polypeptide cleavage is the same as in Example 1.

[0035] (4) The polypeptide was purified in the same manner as in Example 1 to obtain G5P2, the mass spectrum of which is shown below. Figure 2 As shown, the theoretical molecular weight of G5P2 is 2739, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0036] Example 3: Synthesis of antimicrobial peptide 5P2 (1) The activation of the resin is the same as in Example 1.

[0037] (2) The synthesis of Fmoc-5P2-MBHA was carried out by sequentially condensing Leu, Lys, Leu, Lys, Leu, Lys, Leu, Pro, Lys, Leu, Lys, Leu, Lys, Pro, Leu, Lys, Leu, Lys, Leu, Lys, Leu, to obtain Fmoc-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Leu-MBHA.

[0038] (3) The polypeptide cleavage is the same as in Example 1.

[0039] (4) The polypeptide was purified in the same manner as in Example 1, yielding 5P2, the mass spectrum of which is shown below. Figure 3 As shown, the theoretical molecular weight calculation result of 5P2 is 2625, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0040] Example 4: Synthesis of antimicrobial peptide G6P2 (1) The activation of the resin is the same as in Example 1.

[0041] (2) The synthesis of Fmoc-G6P2-MBHA was carried out by sequentially condensing Gly, Leu, Lys, Leu, Lys, Leu, Lys, Leu, Lys, Pro, Leu, Lys, Leu, Lys, Leu, Lys, Leu, Lys, Leu, Leu, Pro, Lys, Leu, Lys, Leu, Gly to obtain Fmoc-Gly-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Lys-Leu-Lys-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Lys-Leu-Gly-MBHA.

[0042] (3) The polypeptide cleavage is the same as in Example 1.

[0043] (4) The polypeptide was purified in the same manner as in Example 1 to obtain G6P2, the mass spectrum of which is shown below. Figure 4 As shown, the theoretical molecular weight of G6P2 is 3221, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

[0044] Example 5: Synthesis of antimicrobial peptide G6P3 (1) The activation of the resin is the same as in Example 1.

[0045] (2) The synthesis of Fmoc-G6P3-MBHA was carried out by sequentially condensing Gly, Leu, Lys, Leu, Lys, Leu, Lys, Pro, Leu, Lys, Leu, Lys, Leu, Lys, Pro, Lys, Leu, Lys, Leu, Lys, Leu, Pro, Lys, Leu, Lys, Leu, Leu, Leu, Gly to obtain Fmoc-Gly-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Leu-Lys-Leu-Lys-Leu-Lys-Pro-Lys-Leu-Lys-Leu-Lys-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Pro-Lys-Leu-Lys-Leu-Lys-Leu-Gly-MBHA.

[0046] (3) The polypeptide cleavage is the same as in Example 1.

[0047] (4) The polypeptide was purified in the same manner as in Example 1 to obtain G6P3, the mass spectrum of which is shown below. Figure 5 As shown, the theoretical molecular weight of G6P3 is 3318, which is consistent with the mass spectrometry identification result, proving that the antimicrobial peptide structure is correct.

Claims

1. A group of low-toxicity, broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences, characterized in that, The antimicrobial peptide is expressed via sequence G(LK). n (KL) n Inserting 2-3 Pros at the center and symmetrical positions of G-NH2 yields GnPm; where n is 5 or 6, representing the number of repetitions of LK, and m represents the number of Pros. The Gs at both ends of the sequence can be removed.

2. The group of low-toxicity, broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences as described in claim 1, characterized in that, The low-toxicity broad-spectrum antimicrobial peptide is G4P1, G5P2, 5P2, G6P2, or G6P3. Its amino acid sequences are shown as SEQ ID NO.1-SEQ ID NO.5, respectively.

3. The group of low-toxicity, broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences as described in claim 1, characterized in that, The low-toxicity broad-spectrum antimicrobial peptide is G5P2 or 5P2.

4. The use of the group of low-toxicity broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences as described in any one of claims 1-3 in the preparation of clinical antibacterial drugs, characterized in that, The bacteria inhibited by the low-toxicity broad-spectrum antimicrobial peptide are Gram-positive and / or Gram-negative bacteria.

5. The use of a group of low-toxicity broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences as described in any one of claims 1-3 in the preparation of clinical antimicrobial drugs in combination with antibiotics.

6. The application of the group of low-toxicity broad-spectrum antimicrobial peptides having proline, glycine, and palindromic sequences as described in claim 5 in the preparation of clinical antimicrobial drugs in combination with antibiotics, characterized in that, The antibiotic is Gentamicin, Polymyxin B, or Rifampicin, and the antimicrobial peptide is G5P2 or 5P2.

Images