Antibacterial peptide from red deer as well as preparation method and application of antibacterial peptide
By preparing the sika deer antimicrobial peptide CeP28, the problem of traditional antibiotic resistance has been solved, and it has achieved effective inhibition and sterilization of multidrug-resistant strains and fungi, showing potential as an anti-infective drug.
Patent Information
- Application Number
- CN202511821230.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-03-06
AI Technical Summary
Existing antibiotics face the problem of drug resistance. Traditional antibiotics are ineffective against multidrug-resistant strains, and the development of new drugs is slow. There is an urgent need for new antibacterial drugs to deal with multidrug-resistant strains and fungal infections.
An antimicrobial peptide, CeP28, derived from red deer, was designed and synthesized via solid-phase chemical synthesis. Its amino acid sequence is GRFRRFRKKLGKYIKKAIPFLPLLNLG, and it exhibits broad-spectrum antimicrobial activity and low hemolytic toxicity.
CeP28 exhibits potent inhibitory and bactericidal effects against Gram-negative bacteria, Gram-positive bacteria, and fungi. It also shows low hemolytic activity against erythrocytes and low toxicity to mouse fibroblasts, making it a potential broad-spectrum anti-infective drug.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to an antimicrobial peptide CeP28 derived from red deer, its preparation method, and its application. Background Technology
[0002] Antimicrobial resistance is a major global health threat, causing more than 5 million deaths annually. This alarming number could double by 2025 if new antibiotics are not discovered and developed to curb this phenomenon. The WHO's recently updated list of bacterial pathogens highlights multidrug-resistant Gram-negative bacteria (carbapenem-resistant Acinetobacter baumannii, third-generation cephalosporin-resistant, and carbapenem-resistant Enterobacteriaceae). High-priority pathogens have been identified as fluoroquinolone-resistant Salmonella typhi, non-typhi Salmonella, carbapenem-resistant Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (Cotugno S, De Vita E, Frallonardo L, et al. Antimicrobial Resistance and Migration: Interrelation Between Two Hot Topics in Global Health. Ann Glob Health. 2025; 91(1): 12.).
[0003] Faced with the challenges of traditional antibiotics, antimicrobial peptides have become a research hotspot due to their unique mechanisms of action and low susceptibility to inducing drug resistance. Antimicrobial peptides possess multiple antibacterial mechanisms, capable of physically disrupting bacterial cell membranes and even inducing reactive oxygen species (ROS) generation. This mechanism makes it difficult for bacteria to develop resistance through a single mutation.
[0004] Meanwhile, with the development of artificial intelligence technology, this technology has begun to be widely applied to peptide design. AI-based protein structure design and structure-activity relationship analysis have greatly accelerated the discovery of highly effective and low-toxicity antimicrobial peptides. Summary of the Invention
[0005] Objective of this invention: To address the emergence of antibiotic resistance, this invention provides an antimicrobial peptide, CeP28, derived from red deer, along with its preparation method and applications. CeP28 exhibits broad-spectrum antimicrobial activity, demonstrating potent inhibitory and bactericidal effects against most bacterial pathogens and even some fungi, while showing almost no hemolytic activity against erythrocytes. Therefore, this invention has the potential to be used in the preparation of anti-infective drugs, adding a polypeptide drug to the clinical approach for the elimination of bacteria and fungi.
[0006] Technical solution: The objective of this invention is achieved through the following technical solution:
[0007] This invention provides an antimicrobial peptide derived from red deer, the amino acid sequence of which is shown in SEQ ID No. 1:
[0008] SEQ ID No. 1: GRFRRFRKKLGKYIKKAIPFLPLLNLG.
[0009] The present invention also provides a specific method for preparing the antimicrobial peptide, comprising the following steps:
[0010] Step 1: Using the human-derived cathelicidin family antimicrobial peptide precursor sequence LL-37 (Accession: NP_004336) as a template, the online tool "BLAST" was used on the NCBI website to search the red deer proteome database to obtain the cathelicidin family antimicrobial peptide precursor sequence from this species. This sequence was then imported into MEGA 11 software along with other known cathelicidin family antimicrobial peptide precursor sequences for multiple sequence alignment. The conserved sequence of the Cathlin region was verified. Simultaneously, the amino acid residue cleavage site of the elastase was predicted to determine the mature peptide sequence, yielding CeP28, whose amino acid sequence is shown in SEQ ID No. 1.
[0011] SEQ ID No. 1: GRFRRFRKKLGKYIKKAIPFLPLLNLG.
[0012] Step 2: CeP28 was prepared in vitro using a solid-phase chemical synthesis method.
[0013] Beneficial effects
[0014] The amino acid sequence of the antimicrobial peptide CeP28 in this invention is shown in SEQ ID NO.1. It exhibits good broad-spectrum antimicrobial activity against Gram-negative bacteria, Gram-positive bacteria, and fungi. Furthermore, CeP28 shows low hemolytic activity against sheep erythrocytes and low toxicity against mouse fibroblast L929 cells. Therefore, the antimicrobial peptide CeP28 of this invention can serve as a potential broad-spectrum anti-infective drug. Attached Figure Description
[0015] Figure 1 CeP28 exhibits hemolytic activity against sheep erythrocytes; Figure 2 CeP28 for E. coli ATCC and E. coli CI The bactericidal curves, where A represents the effect of CeP28 on E. coli. CI B is the bactericidal curve of CeP28 against E. coli O157, and C is the bactericidal curve of CeP28 against E. coli ATCC 25922. Figure 3For CeP28 to S. aureus ATCC and S. aureus CI The bactericidal curves, where A represents the effect of CeP28 on S. aureus. C1I The bactericidal curves, B represents CeP28 against S. aureus. C12 The bactericidal curves are shown, where C is the bactericidal curve of CeP28 against S. aureus ATCC 25927; Figure 4 The bactericidal curve of CeP28 against S. typhimurium SL 1344; Figure 5 The toxicity of CeP28 to mouse fibroblast L929 cells. Detailed Implementation
[0016] The technical solution of the present invention will be described in detail below through specific embodiments, but the scope of protection of the present invention is not limited to the embodiments described.
[0017] The polypeptides designed in this invention were directly synthesized by Jier Biochemical (Shanghai) Co., Ltd.
[0018] Example 1: Design and Synthesis of Antimicrobial Peptide CeP28
[0019] Using the human-derived cathelicidin family antimicrobial peptide precursor sequence LL-37 (Accession: NP_004336) as a template, the proteome database of red deer (Cervus elaphus) was searched using the online tool "BLAST" on the NCBI website to obtain the cathelicidin family antimicrobial peptide precursor sequence from this species. This sequence, along with other known cathelicidin family antimicrobial peptide precursor sequences, was imported into MEGA 11 software for multiple sequence alignment. The conserved sequence of the Cathlin region was verified. Simultaneously, the amino acid residue cleavage site of elastase was predicted, and the mature peptide sequence was determined, yielding CeP28, whose amino acid sequence is shown in SEQ ID No. 1, specifically: GRFRRFRKKLGKYIKKAIPFLPLLNLG.
[0020] Example 2: Determination of the in vitro antimicrobial activity of antimicrobial peptides
[0021] 1. Determination of the minimum inhibitory concentration (MIC) of the antimicrobial peptide CeP28 against bacteria:
[0022] Following the standard serial dilution method, CeP28 was prepared into a series of concentration gradients (1, 2, 4, 8, 16, 32, 64, 128 μg / ml) using the corresponding liquid culture medium. This was then compared with a bacterial concentration adjusted to 2 × 10⁻⁶ using the same liquid culture medium. 5Equal volumes of the test bacterial solutions at CFU / ml were mixed. The lowest drug concentration at which no bacterial growth was observed visually was defined as the MIC. The results are shown in Table 1. (The strains tested in this example included Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Salmonella typhimurium, Salmonella enteritidis, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and Cryptococcus neoformans.) (neoformans). The positive control drugs used in this example include oxociprofloxacin, ceftazidime, and penicillin (all positive control drugs were from Aladdin Company; the clinical strains were obtained from clinical isolation in the hospital, and the standard strains were purchased from Guangdong Provincial Center for Microbial Culture Collection).
[0023] Table 1: In vitro antimicrobial activity (MIC) of antimicrobial peptide CeP28
[0024]
[0025] Note: CI refers to strains obtained clinically.
[0026] 2. Bactericidal curves of antimicrobial peptide CeP28 against different bacteria
[0027] The bacterial suspension was resuspended in LB medium and the bacterial concentration was adjusted to 10. 5 For each CFU / ml strain, 2 mL was taken and placed in a sterile glass tube. A specific volume of each drug was added, and the strains were treated separately (S. typhimurium: one group received CeP28, the other received Oxociprofloxacin; E. coli: one group received CeP28, the other received Ceftazidime; S. aureus: one group received CeP28, the other received Penicillin). The volume was adjusted with blank medium to obtain a final drug concentration of 4×MIC in 3 mL. A blank control system without added drugs was also included.
[0028] CeP28, Oxociprofloxacin, Ceftazidime, and Penicillin were directly diluted to the required concentrations using LB liquid medium.
[0029] Each experimental group was incubated at 37℃. At 0, 0.5, 1, 2, 4, 8, 12, and 24 hours after incubation, 100 μl of the mixed culture was taken out and serially diluted with sterile physiological saline. 200 μl of each diluted culture was then placed in sterile Petri dishes, and approximately 10 ml of LB solid medium at approximately 50℃ was added. The mixture was shaken thoroughly, cooled, and incubated at 37℃ for 36 hours. Finally, viable cell counts were performed, and a bactericidal curve was plotted with drug action time on the x-axis and colony count on the y-axis. The results are shown below. Figure 2-4 As shown.
[0030] This experiment selected plates with colony counts between 30 and 300, and the gradient settings for each experimental group were as follows:
[0031] For the control group and the positive control group, three dilutions of 1:1000, 1:10000, and 1:100000 were used for plate pouring at 0, 0.5, 1, 2, and 4 hours before the positive control group. After 4 hours, dilutions of 1:100, 1:1000, and 1:10000 were used for plate pouring. For the CeP28 group, three dilutions of 1:100, 1:1000, and 1:10000 were used at 0, 0.5, and 1 hours. At 4, 8, 12, and 24 hours, 200 μl of undiluted stock solution and dilutions of 1:10 and 1:100 were used for plate pouring.
[0032] The sterilization curve results showed that, compared with 0 h, CeP28 significantly reduced the levels of *S. typhimurium* SL1344, *E. coli* O157, *S. aureus* ATCC 25927, and *S. aureus* at 1 h and 2 h. CI1 S. aureus CI2 E. coli ATCC25922, E. coli CI The number of live bacteria is high, and the bacteria mentioned above can be significantly eliminated within 4 hours.
[0033] 3. Hemolytic activity of antimicrobial peptide CeP28 against sheep erythrocytes
[0034] Take 3 ml of fresh, sterile, defibrinated sheep blood (Nanjing Senbeijia Biotechnology) into a sterile EP tube, centrifuge at 3000 rpm for 10 min, carefully discard the supernatant, and resuspend in PBS and wash three times. Add PBS to the precipitate to prepare a 3% red blood cell suspension, and add 50 μl / well to a 96-well plate. Then add an equal volume of peptide solution to each well to achieve a final peptide concentration of 4, 8, 16, 32, 64, and 128 μg / ml. Red blood cell suspension treated with 1% Triton X-100 serves as a positive control, and red blood cell suspension treated with PBS serves as a negative control. The results are compared with melitin at the same concentration gradient, with three replicates per group. Incubate at 37℃ for 1 h. Centrifuge at 3000 rpm for 10 min, collect the supernatant and place it in another sterile 96-well plate, and measure the absorbance at 450 nm. The positive control was set at 100% hemolysis rate, and the negative control was set at 0% hemolysis rate. The hemolysis rate of the polypeptide on red blood cells was calculated using the following formula:
[0035]
[0036] The results are as follows Figure 1 As shown.
[0037] The results of hemolytic activity showed that when the concentration of CeP28 reached 64×MIC, the hemolysis rate on sheep erythrocytes was still less than 5%, indicating very low hemolytic activity.
[0038] 4. Toxicity of the antimicrobial peptide CeP28 to mouse fibroblast L929 cells
[0039] When the density of mouse fibroblast L929 cells reached approximately 80%–90%, they were digested, centrifuged, and the supernatant was removed. The cells were then resuspended in DMEM complete medium and diluted to a concentration of 5–10 × 10⁻⁶ cells / mL. 4 Cells / ml. Add 100 μl of cell suspension to each well of a 96-well plate and incubate for 24 h. After cell attachment, aspirate the culture supernatant from the wells, add 100 μl of culture medium containing different drug concentrations, and incubate at 37°C for 24 h with 5% CO2. After incubation, add 10 μl of MTT solution (5 mg / ml) to each well, continue incubating for 4 h, centrifuge at 1000 rpm for 10 min, carefully aspirate the supernatant, add 100 μl of dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10 min to fully dissolve the crystals. Analyze the OD value using an ELISA reader. 490 Measure the absorbance of each well and calculate the cell viability using the following formula:
[0040]
[0041] The blank group refers to an equal volume of DMEM complete culture medium without any drugs.
[0042] The results are as follows Figure 5 As shown.
[0043] The results of the MTT assay showed that CeP28 had low toxicity to mouse fibroblast L929 cells, and its median lethal concentration (LD50) was much lower than the concentration at which it could effectively exert its antibacterial activity.
[0044] amino acid sequence:
[0045] SEQ ID No. 1: GRFRRFRKKLGKYIKKAIPFLPLLNLG.
Claims
1. An antibacterial peptide CeP28 derived from Cervus elaphus, characterized in that, The amino acid sequence of which is shown as SEQ ID No.
1.
2. A pharmaceutical composition, characterized by, The amino acid sequence of which is shown as SEQ ID No.
1.
3. The use of the antibacterial peptide CeP28 of claim 1 or the pharmaceutical composition of claim 2 in the preparation of an anti-infective drug.
4. Use according to claim 3, characterized in that, The bacteria include any one or more of gram-negative bacteria, gram-positive bacteria and fungi.
5. Use according to claim 4, characterized in that, The gram-negative bacteria include any one or more of Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella and Acinetobacter baumannii; the gram-positive bacteria include any one or more of Staphylococcus aureus and Staphylococcus epidermidis; and the fungi include any one or more of Candida albicans and Cryptococcus neoformans.