Scylla antibacterial peptide Sptesin 135-154 and application thereof
By developing the blue crab antimicrobial peptide Sptesin135-154, the problems of single target and toxic side effects of existing antimicrobial drugs have been solved, and efficient inhibition and killing of a variety of bacteria and fungi have been achieved, especially showing excellent effects in high-temperature processing and biofilm infection scenarios, and is suitable for medical treatment and aquaculture.
Patent Information
- Application Number
- CN202511057395.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-10-17
AI Technical Summary
Existing antimicrobial drugs have a single target, are prone to drug resistance, have toxic side effects, have long treatment cycles and high costs, are unable to effectively deal with complex infections such as biofilm-related infections, and cannot meet increasingly severe clinical needs.
A 20-amino acid blue crab antimicrobial peptide, Sptesin135-154, has been developed. It is cationic, hydrophobic, and highly water-soluble. It has broad-spectrum resistance to bacterial and fungal infections by destroying microbial cell membranes and inhibiting biofilm formation.
It exhibits significant antibacterial and bactericidal effects, has high efficiency in inhibiting and killing a variety of bacteria and fungi, has good thermal stability and low cytotoxicity, is suitable for medical treatment and aquaculture, and solves the problems of drug resistance and toxic side effects of existing drugs, especially showing excellent effects in high-temperature processing and biofilm infection scenarios.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of marine molecular biology, and particularly relates to a blue crab antibacterial peptide Sptesin 135-154 and application thereof. BACKGROUND
[0002] Bacterial and fungal infections are major challenges in global public health, with incidence rates increasing in recent years driven by factors such as population aging, increased use of immunosuppressive therapies, and climate change affecting the ecological distribution of pathogens. Common pathogenic bacteria include Gram-positive bacteria (such as Enterococcus faecium and Enterococcus faecalis) and Gram-negative bacteria (such as Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Vibrio fluvialis, and Vibrio harveyi), which have high genetic diversity and adaptability and can evade host immunity and drug effects by forming biofilms or mutating. Fungal infections are also severe, with common pathogenic fungi including Candida albicans, Pichia pastoris, Cryptococcus neoformans, Candida krusei, Candida tropicalis, Fusarium solani, Fusarium graminearum, and Aspergillus ochraceus, which can cause systemic infections, especially in immunodeficient patients. In recent years, the emergence of drug-resistant strains has exacerbated the problem, such as bacterial multi-drug resistance to methicillin and carbapenems, and fungal resistance to fluconazole and amphotericin B, leading to increased failure rates in clinical treatment and heavier medical burden.
[0003] Currently, commonly used clinical drugs include penicillins, cephalosporins, macrolide antibiotics, and azole and polyene antifungal drugs. These drugs exert their effects by inhibiting bacterial cell wall synthesis, protein synthesis, or fungal cell membrane function, to some extent controlling infections. However, existing technologies have significant limitations. First, the single drug target is easily evaded by pathogens through genetic mutation or acquired resistance mechanisms, leading to rapid spread of drug-resistant strains. Second, many drugs have strong toxic side effects, such as nephrotoxicity, hepatotoxicity, and neurotoxicity, especially in special populations such as children, pregnant women, and the elderly. In addition, the treatment period is long, the cost is high, and the effect is limited in complex infections (such as biofilm-related infections), which cannot meet the increasingly severe clinical needs. The World Health Organization (WHO) has listed antimicrobial resistance as one of the top ten global health threats, calling for the development of new anti-infective drugs to address this crisis.
[0004] As a kind of natural small molecule polypeptide, antimicrobial peptides (AMPs) are widely present in animals, plants and microorganisms, with broad-spectrum antibacterial activity, low toxicity and low drug resistance risk, and are regarded as a potential substitute for traditional antibiotics. The main mechanisms of action of antimicrobial peptides include destroying microbial cell membranes, interfering with metabolic processes and inhibiting biofilm formation. These multi-target effects make it difficult for pathogens to develop drug resistance. Marine organisms, especially crustaceans, are an important resource for mining new antimicrobial peptides due to their unique living environment and developed natural immune defense system. Scylla paramamosain, as an important economic crab, may contain a variety of polypeptides with antibacterial potential. Existing studies have identified some antimicrobial peptides from Scylla paramamosain, but the comprehensive research on broad-spectrum antibacterial and fungal activity, no cytotoxicity and practical application (such as medical, food and aquaculture) is still insufficient. The existing antimicrobial peptides have optimization space in terms of thermal stability, antibiofilm effect and cell safety, and cannot fully meet the clinical and industrial needs. Therefore, it is necessary to further explore new antimicrobial peptides to overcome the limitations of traditional drugs and provide safer and more effective anti-infection solutions. SUMMARY
[0005] The present application aims to overcome the defects of the prior art and provide a rare earth synergistic non-intumescent ultra-thin fireproof and thermal insulation coating.
[0006] Another object of the present application is to provide a preparation method of the above-mentioned rare earth synergistic non-intumescent ultra-thin fireproof and thermal insulation coating.
[0007] The technical scheme of the present application is as follows:
[0008] A scylla paramamosain antibacterial peptide Sptesin 135-154 , and the amino acid sequence is shown as SEQ ID NO. 01.
[0009] The above-mentioned scylla paramamosain antibacterial peptide Sptesin 135-154 application in preparing antibacterial compositions.
[0010] In a preferred embodiment of the present application, the antibacterial composition has inhibitory and killing effects on Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Vibrio fluvialis and Vibrio harveyi.
[0011] An antibacterial composition, the effective component of which comprises the above-mentioned scylla paramamosain antibacterial peptide Sptesin 135-154 .
[0012] In a preferred embodiment of the present application, the effective component is the above-mentioned scylla paramamosain antibacterial peptide Sptesin135-154 .
[0013] The above-mentioned blue crab antibacterial peptide Sptesin 135-154 The application in preparing an antifungal composition.
[0014] In a preferred embodiment of the present application, the antifungal composition has inhibitory and killing effects on Candida albicans, Pichia pastoris, Cryptococcus neoformans, Candida krusei, Candida tropicalis, Fusarium solani, Fusarium graminearum and Aspergillus ochraceus.
[0015] An antifungal composition, the effective component of which comprises the above-mentioned blue crab antibacterial peptide Sptesin 135-154 .
[0016] In a preferred embodiment of the present application, the effective component is the above-mentioned blue crab antibacterial peptide Sptesin 135-154 .
[0017] The present application has the following beneficial effects:
[0018] 1. The present application is composed of 20 amino acids, with a molecular weight of 2250.79 Dalton, containing 2 arginines and 3 lysines. HeliQuest predicts that the antibacterial peptide has a charge number of +5 and a hydrophobicity of 34.8%, which is a cationic short peptide with good water solubility, high antibacterial activity, wide antibacterial spectrum and safety, and has a broad application prospect.
[0019] 2. The present application shows significant inhibitory and killing effects on various bacteria (such as Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Vibrio fluvialis, Vibrio harveyi) and fungi (such as Candida albicans, Pichia pastoris, Cryptococcus neoformans, Candida krusei, Candida tropicalis, Fusarium solani, Fusarium graminearum and Aspergillus ochraceus). The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC / MFC) experimental results show that the MIC of most pathogens is in the range of 0-48 μM, and the killing efficiency is high, which provides an effective means for controlling drug-resistant infections.
[0020] 3. The present application can kill 99.99% of Candida albicans within 180 minutes at a concentration of 24 μM, and the bactericidal kinetics curve shows that it acts rapidly, which is superior to some traditional drugs.
[0021] 4. The present application can still effectively inhibit the growth of Candida albicans after being heated at 100 ℃ for 30 minutes, and is suitable for high-temperature processing scenes, such as food preservation or pharmaceutical preparations.
[0022] 5. The present application significantly inhibits the formation of Candida albicans biofilm at a concentration of 6-24 μM, solving the problem of poor effect of traditional drugs on biofilm infection.
[0023] 6. MTS assay showed that the present invention had no cytotoxicity to human kidney epithelial cells (HEK-293T) and zebrafish embryonic cells (ZF4), and the cell survival rate was close to 100% at high concentrations, indicating that it has excellent biosafety and is suitable for medical treatment and aquaculture.
[0024] 7. The present invention is a cationic short peptide (charge +5, hydrophobicity 34.8%) with good water solubility. It can be used to prepare antibacterial / fungal compositions and has potential applications in medical infection treatment, food preservation, and aquaculture disease prevention and control, meeting the needs of green and sustainable development. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 It shows that the antimicrobial peptide Sptesin in Example 3 of the present invention 135-154 Bactericidal kinetic curve against Candida albicans CGMCC 2.2411.
[0026] Figure 2 It shows that the antimicrobial peptide Sptesin in Example 4 of the present invention 135-154 Thermal stability curve of Candida albicans CGMCC 2.2411.
[0027] Figure 3 It shows that the antimicrobial peptide Sptesin in Example 5 of the present invention 135-154 Results of antibiofilm activity assay against Candida albicans CGMCC 2.2411.
[0028] Figure 4 The MTS method for detecting the antimicrobial peptide Sptesin in Example 6 of the present invention is shown. 135-154 The results of the cytotoxicity test on HEK-293T and ZF4 cells, where the horizontal axis is Sptesin 135-154 The vertical axis represents protein concentration (μM), and the vertical axis represents cell survival rate (%). DETAILED DESCRIPTION
[0029] The technical solution of the present invention is further illustrated and described below through specific implementation methods in conjunction with the accompanying drawings.
[0030] Example 1: Scylla antimicrobial peptide Sptesin 135-154 Preparation
[0031] The blue crab antimicrobial peptide Sptesin in this example 135-154 The amino acid sequence is SEQ ID NO.01: RGYRLALLGNKGITMMLKKS.
[0032] In this example, Nanjing GenScript Co., Ltd. was commissioned to synthesize the blue crab antimicrobial peptide Sptesin with a purity of more than 95%.135-154 and provide polypeptide molecular weight, HPLC detection information, use HeliQuest to predict its charge and hydrophobicity, and other physicochemical parameters are predicted by ProtParam, antibacterial peptide Sptesin 135-154 The physicochemical parameters are shown in Table 1.
[0033] Table 1 antibacterial peptide Sptesin 135-154 Physicochemical parameters
[0034]
[0035] Example 2 Blue crab antibacterial peptide Sptesin 135-154 Determination of minimum bactericidal concentration
[0036] In this example, the blue crab antibacterial peptide Sptesin obtained in Example 1 135-154 was determined for minimum bactericidal concentration, and the strains involved were: Candida albicans, Pichia pastoris, Cryptococcus neoformans, Candida krusei, Candida tropicalis, Fusarium solani, Fusarium graminearum, Aspergillus ochraceus, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Vibrio fluvialis and Vibrio harveyi purchased from the Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences.
[0037] The specific method is as follows:
[0038] (1) Activate the strains, streak the preserved bacteria on nutrient broth plates, spread the Vibrio on 2216 plates, streak the yeast fungi on YPD plates, and spread the molds on potato dextrose plates, and incubate at 37°C or 28°C for 1-7 days.
[0039] (2) Pick colonies from each plate and inoculate in the corresponding liquid medium, and incubate overnight in a constant temperature shaker.
[0040] (3) Centrifuge to collect the bacterial cells, resuspend the bacterial cells with sterile 10 mM sodium phosphate buffer (pH = 7.4), then dilute the bacteria with MH liquid medium to a final concentration of about 5 × 10 5 CFU / mL, dilute Vibrio with TSB + 2% NaCl liquid medium, and dilute fungi with RPMI-MOPS liquid medium to a final concentration of 5 × 10 4 CFU / mL.
[0041] (4) Dissolve the synthesized Sptesin 135-154 powder in sterile Milli-Q water, filter with a 0.22 μm filter, and dilute the protein concentration by 6 μM, 12 μM, 24 μM, 48 μM, 96 μM, and 192 μM.
[0042] (5) In 96-well cell culture plates, set up blank control group, negative control group and test experimental group for each bacteria to be tested, and set up three parallels for each group:
[0043] a Blank control group: 50 μL of the protein sample to be tested (Sptesin 135-154 ) and 50 μL of the culture medium of the diluted bacteria.
[0044] b Negative control group: 50 μL of sterile Milli-Q water and 50 μL of bacterial suspension.
[0045] c Test experimental group: 50 μL of the protein sample to be tested (Sptesin 135-154 ) and 50 μL of bacterial suspension.
[0046] Place the 96-well cell culture plates in a 37°C or 28°C incubator and incubate for 1-2 days. Observe and record the minimum inhibition concentration (MIC) results on bacteria. Take an appropriate amount of bacterial solution and drop it on the corresponding solid culture medium plate, and incubate at the appropriate temperature for 1-2 days. Observe and record the minimum bactericidal concentration (MBC) on bacteria and the minimum fungicidal concentration (MFC) on fungi.
[0047] The blue crab antibacterial peptide Sptesin 135-154 obtained in this example has a minimum bactericidal concentration as shown in Table 2, indicating that Sptesin 135-154 has broad-spectrum antibacterial activity.
[0048] Table 2 Antimicrobial activity of blue crab antibacterial peptide Sptesin 135-154
[0049]
[0050]
[0051] Example 3 Bactericidal kinetics curve of blue crab antibacterial peptide Sptesin 135-154
[0052] This example determines the bactericidal kinetics curve of the blue crab antibacterial peptide Sptesin 135-154 obtained in Example 1, and the strain involved is Candida albicans CGMCC 2.2411.
[0053] The specific method is as follows:
[0054] The antibacterial test method is consistent with the minimum bactericidal concentration determination method in Example 1. After the antimicrobial peptide and Candida albicans are co-incubated for a certain period of time, the co-incubated mixture is diluted gradiently and then coated on a plate. After static incubation, the colonies are counted.
[0055] Scylla antimicrobial peptide Sptesin 135-154 The bactericidal kinetic curve of Candida albicans CGMCC 2.2411 is as follows Figure 1 As shown, 24 μM Sptesin 135-154 It can kill 99.99% of Candida albicans in 180 minutes.
[0056] Example 4: Scylla antimicrobial peptide Sptesin 135-154 Thermal stability
[0057] This example is to compare the blue crab antimicrobial peptide Sptesin obtained in Example 1 135-154 The thermal stability test was conducted using the strain Candida albicans CGMCC 2.2411.
[0058] The specific method is as follows:
[0059] The specific method is similar to the antibacterial activity determination in Example 2. 135-154 To a final concentration of 1 times MFC, heat in boiling water at 100°C for 10 min, 20 min, 30 min and 60 min, and place on ice for later use. 135-154 Incubate with the test bacteria for 48 hours and continuously monitor OD using a microplate reader 600 value.
[0060] Scylla antimicrobial peptide Sptesin 135-154 The thermal stability results of Candida albicans CGMCC 2.2411 are as follows Figure 2 As shown, Sptesin 135-154 After continuous heat treatment at 100℃ for 30 minutes, the antimicrobial peptide still effectively inhibited the growth of Candida albicans, indicating that it has strong thermal stability.
[0061] Example 5: Scylla antimicrobial peptide Sptesin 135-154 Anti-biofilm activity
[0062] This example is to compare the blue crab antimicrobial peptide Sptesin obtained in Example 1 135-154 The anti-biofilm activity test was conducted using the Candida albicans CGMCC 2.2411 strain.
[0063] The specific method is as follows:
[0064] According to the antibacterial test method, the concentration of the bacterial solution was adjusted to 5×10 4CFU / mL, and placed on ice for standby. The crab antibacterial peptide Sptesin 135-154 was added to the 96-well culture plate to a final concentration of 1 times MFC, mixed with an equal volume of bacterial solution, and then incubated in a 28°C incubator for 48 h to form a biofilm. The supernatant was discarded, and the cells were washed twice with PBS to remove unbound bacteria.
[0065] 200 μL of 0.1% crystal violet solution was added to each well, and the plate was stained at room temperature for 15 min. The solution was then discarded, and the wells were washed three times with distilled water. After the plate was dried, 200 μL of 95% ethanol was added to each well to dissolve the crystal violet, and the OD 600 value was measured using a microplate reader. The lower the absorbance value, the less biofilm formed, and the antibacterial peptide was evaluated for its inhibitory effect on the early stage of biofilm formation.
[0066] Crab antibacterial peptide Sptesin 135-154 was added to the 96-well culture plate to a final concentration of 1 times MFC, mixed with an equal volume of bacterial solution, and then incubated in a 28°C incubator for 48 h to form a biofilm. The supernatant was discarded, and the cells were washed twice with PBS to remove unbound bacteria. Figure 3 135-154 The results showed that Sptesin 135-154 had a certain inhibitory effect on the biofilm formation of C. albicans at 6 μM, and the inhibitory effect was more significant at 24 μM.
[0067] Example 6: MTS method for detecting and evaluating the cytotoxicity of crab antibacterial peptide Sptesin 135-154
[0068] In this example, human kidney epithelial cells (HEK-293T) and zebrafish embryonic cells (ZF4) were selected to determine the cytotoxicity of crab antibacterial peptide Sptesin 5
[0069] The specific method is as follows:
[0070] (1) Collect cells in good growth condition, and adjust the concentrations of HEK-293T and ZF4 cells to 10 5 individuals / mL, respectively. Add 100 μL of the above cell suspension to each well of a 96-well cell culture plate, and place the cells in a cell incubator with 5% CO2.
[0071] (2) Remove the culture medium, and add culture medium containing different concentrations of Sptesin 135-154 , and incubate for 24 h.
[0072] (3) Add 20 μL of MTS-PMS mixed solution, and incubate for 4 h. Then, read the plate using a microplate reader, and calculate the cell survival rate. 492nm
[0073] The results are shown in the following table: Figure 4 135-154 Protein concentration (μM), ordinate cell survival rate (%) indicating Sptesin 135-154 No cytotoxicity on HEK-293T and ZF4 cells.
[0074] The above description is only the preferred embodiment of the present application, and therefore cannot limit the scope of the present application. Any equivalent changes and modifications made according to the scope and content of the present patent should still be within the scope of the present application.
Claims
1. A blue crab antimicrobial peptide Sptesin 135-154 , characterized in that: Its amino acid sequence is shown in SEQ ID NO.
01.
2. The blue crab antimicrobial peptide Sptesin according to claim 1 135-154 Application in the preparation of antibacterial compositions.
3. The use according to claim 2, characterized in that: The antibacterial composition has inhibitory and killing effects on Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Acinetobacter baumannii, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas fluorescens, Vibrio fluviatilis and Vibrio harveyi.
4. An antibacterial composition, characterized in that: Its active ingredient includes the blue crab antimicrobial peptide Sptesin according to claim 1 135-154 .
5. The antibacterial composition according to claim 4, wherein: The active ingredient is the blue crab antibacterial peptide Sptesin according to claim 1 135-154 .
6. The blue crab antimicrobial peptide Sptesin according to claim 1 135-154 Application in the preparation of antifungal compositions.
7. The use according to claim 6, characterized in that: The antifungal composition has inhibitory and killing effects on Candida albicans, Pichia pastoris, Cryptococcus neoformans, Candida krusei, Candida tropicalis, Fusarium solani, Fusarium graminearum and Aspergillus ochraceus.
8. An antifungal composition, characterized in that: Its active ingredient includes the blue crab antimicrobial peptide Sptesin according to claim 1 135-154 .
9. The antifungal composition according to claim 8, wherein: The active ingredient is the blue crab antibacterial peptide Sptesin according to claim 1 135-154 .