Antibacterial peptide pr-7 and uses thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses an antibacterial peptide PR-7 and application thereof, and specifically comprises the antibacterial peptide PR-7, an amino acid sequence of which is shown as SEQ ID NO. 1, which has stable bacteriostatic activity on Escherichia coli, and the minimum bacteriostatic concentration is 64 μM. The antibacterial peptide plays an antibacterial role by destroying the bacterial cell membrane structure, is not easy to induce drug resistance of bacteria, has excellent characteristics of high cell selectivity, and reduces the technical problems of prominent antibiotic drug resistance of existing traditional antibiotics and insufficient safety of existing antibacterial peptides. The application can be used for preparing antibacterial agents, antibacterial infection drugs and antibacterial compositions, and can also be used as a bacteriostatic additive in the field of food preservation, and has good industrial application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the antimicrobial peptide PR-7 and its applications. Background Technology

[0002] With accelerating globalization and continuous population growth, food safety issues are becoming increasingly serious, and the incidence of foodborne illnesses is rising significantly. Statistics show that more than 600 million cases of foodborne illness infection occur globally each year, posing a significant threat to public health. Among these, Escherichia coli (E. coli) is a major culprit. Escherichia coli , E. coli It can cause a variety of serious diseases in the human body, ranging from mild diarrhea and gastroenteritis to hemolytic uremic syndrome and sepsis. It poses a particularly prominent health threat to infants, the elderly and people with weakened immune systems.

[0003] In the current healthcare system, traditional antibiotics are the core treatment for bacterial infections. However, the long-term and improper use of antibiotics has led to a continuous exacerbation of bacterial resistance. The emergence of multidrug-resistant bacteria and superbugs has brought unprecedented challenges to the clinical treatment of bacterial infections. At the same time, consumers are increasingly concerned about the safety of chemical preservatives and artificial additives in food, and the demand for natural, safe, and efficient alternatives to food preservation is becoming increasingly urgent.

[0004] Antimicrobial peptides ( Antimicrobial Peptides Antimicrobial peptides (AMPs) are a class of small molecule peptides widely present in the innate immune system of organisms. As natural antimicrobial agents, they possess core advantages such as broad antimicrobial spectrum, strong antimicrobial activity, unique mechanism of action, and low likelihood of inducing bacterial resistance, making them a popular research direction for replacing traditional antibiotics and chemical preservatives. However, most of the currently discovered antimicrobial peptides still have significant drawbacks: some antimicrobial peptides have limited antibacterial activity and excessively high minimum inhibitory concentrations (MICs); some antimicrobial peptides exhibit high cytotoxicity and hemolytic activity, resulting in insufficient biosafety; and some antimicrobial peptides are physicochemically unstable and easily inactivated under physiological conditions, severely limiting their industrial application in the pharmaceutical and food fields. Therefore, developing novel, highly efficient, and highly selective antimicrobial peptides has significant theoretical and practical application value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the antimicrobial peptide PR-7 and its applications. This antimicrobial peptide exhibits excellent antibacterial activity against Escherichia coli, has a unique mechanism of action, and possesses the advantage of high cell selectivity. It can be safely and widely applied in the development of antimicrobial-related products in the fields of medicine and food.

[0006] To achieve the above objectives, the present invention provides an antimicrobial peptide PR-7, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] The application of the antimicrobial peptide PR-7 in the preparation of antimicrobial agents is also provided.

[0008] Preferably, the antibacterial agent is used to inhibit Escherichia coli.

[0009] Preferably, the minimum inhibitory concentration of the antimicrobial peptide PR-7 against Escherichia coli is 64 μM.

[0010] It also provides the application of the antimicrobial peptide PR-7 in the preparation of drugs against Escherichia coli.

[0011] A pharmaceutical composition is also provided, wherein the antimicrobial peptide PR-7 is the main component.

[0012] Preferably, the pharmaceutical composition is an injection, oral preparation, topical preparation, spray, or lyophilized preparation.

[0013] An antimicrobial additive is also provided, which includes the antimicrobial peptide PR-7, and is added to food as an additive.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The antimicrobial peptide PR-7 of this invention was confirmed as a highly active antimicrobial peptide by prediction and verification through a multi-platform bioinformatics system. Among them, the MULTIPEP platform predicted its antimicrobial activity probability as high as 0.950, and the CAMP3 database multi-model prediction core support vector machine model determined that it was an antimicrobial peptide with a probability of 0.741, which clearly demonstrated its excellent antimicrobial potential.

[0015] (2) The antimicrobial peptide PR-7 of the present invention has excellent antimicrobial activity against Escherichia coli, with a minimum inhibitory concentration of 64 μM and a significant concentration-dependent antimicrobial activity. It can achieve high-efficiency inhibition of Escherichia coli within 8 hours. Under the same test conditions, its antimicrobial effect is significantly better than that of similar candidate peptides, and it has high-efficiency antimicrobial ability.

[0016] (3) The antimicrobial peptide PR-7 of the present invention has excellent physicochemical properties and meets the core requirements for drug development. Its molecular weight is moderate, its strong alkalinity is suitable for bacterial cell membrane targets, and it has both suitable hydrophilicity and hydrophobicity, and good stability under physiological conditions; at the same time, its Boman index is in the optimal range of 0-2, which has low protein binding potential and can maintain high bioavailability.

[0017] (4) The antimicrobial peptide PR-7 of the present invention has a wide range of applications. It can be used as an active ingredient to prepare antimicrobial drugs and antimicrobial compositions for treating bacterial infections, and can also be used as a natural antimicrobial additive in the field of food preservation, replacing traditional chemical preservatives. It has both safety and high efficiency and has broad prospects for industrial application.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 This invention illustrates the effects of different candidate antimicrobial peptides at a concentration of 1024 μM on the efficacy of various antimicrobial peptides. E. coli A comparison chart of antibacterial rates, where * represents... p <0.05; Figure 2 In this embodiment of the invention, PR-7 was used to treat various concentrations 8 hours after the treatment. E. coli Antibacterial activity curve; Figure 3 The effects of different concentrations of antimicrobial peptide PR-7 treated in the embodiments of the present invention on the [effects / conditions] E. coli Bactericidal activity (MBC) plate validation. Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0024] Example 1 Bioinformatics analysis and safety evaluation of antimicrobial peptide PR-7.

[0025] This embodiment systematically predicts and analyzes the physicochemical properties, antibacterial activity, and drug-likeness-related indicators of the target antimicrobial peptide PR-7, providing theoretical support for subsequent in vitro activity verification.

[0026] The antibacterial activity of peptides was predicted using the online software CAMPR3. Physicochemical properties of the peptides, including net charge, GRAVY total average hydrophilicity, Wimley-White total residue hydrophobicity scale, and Boman index, were calculated using the online software APD6. Isoelectric point and instability index were predicted using ProtParam in ExPASy.

[0027] Based on the above indicators, the antimicrobial peptide PR-7 (as shown in SEQ ID NO.1) has a Boman index in the range of 0-2, low surface protein binding potential, high bioavailability in vivo, and good drug-like properties; net charge number > 0; GRAVY index < 0, indicating that it has both hydrophilicity and water solubility, good dispersibility under physiological conditions, and a molecular weight of 1811.194 Da; the total hydrophobicity is 43% (as shown in Table 1).

[0028] Table 1. Predicted results of physicochemical properties and antibacterial activity of antimicrobial peptide PR-7

[0029] Based on the CAMP3 database, the antimicrobial activity of target peptides was analyzed using multiple model prediction results to determine whether they are antimicrobial peptides and to calculate the probability of them being antimicrobial peptides.

[0030] The multi-model prediction results are shown in Table 2. The core prediction model, Support Vector Machine (SVM), identified PR-7 as an antimicrobial peptide (AMP) with a probability of 0.741. The ensemble model, Random Forest (RF), also identified it as an antimicrobial peptide with a probability of 0.5545. Based on the combined results of the multi-model analysis, the core model determined PR-7 to be a highly active antimicrobial peptide with clear antimicrobial potential.

[0031] Table 2. Multi-model prediction results of antimicrobial activity of antimicrobial peptide PR-7 from the CAMP3 database.

[0032] Multi-platform cross-validation: The antibacterial activity, family affiliation, and physicochemical properties of the target peptide PR-7 were cross-validated and supplemented using the APD3, MULTIPEP, and DBAAP platforms.

[0033] The results are shown in Table 3. The APD3 database identification results show that the antimicrobial peptide PR-7 is a known member of the antimicrobial peptide family. MULTIPEP activity prediction shows that its antimicrobial activity probability is as high as 0.950, and its antimicrobial and antifungal activity probabilities are 0.472 and 0.378, respectively, confirming that the antimicrobial peptide PR-7 has antimicrobial activity as its core advantage and possesses broad-spectrum antimicrobial potential. In terms of physicochemical properties, DBAAP analysis shows that its molecular weight is 1811.20 Da, its isoelectric point is 10.30 (strongly basic), and its normalized hydrophobicity is -0.24 (slightly hydrophilic), which is consistent with the aforementioned physicochemical property test results. It fully conforms to the typical structure and physicochemical characteristics of cationic antimicrobial peptides, providing a theoretical basis for subsequent in vitro activity verification and drug development studies.

[0034] Table 3. Results of multi-platform cross-validation analysis of antimicrobial peptide PR-7

[0035] In summary, the antimicrobial peptide PR-7 meets the core characteristics of a highly efficient cationic antimicrobial peptide. Validated through multi-platform and multi-model predictions, it possesses extremely high antimicrobial activity potential, excellent physicochemical properties, and good drug-likeness, providing a sufficient theoretical basis for subsequent in vitro antimicrobial activity validation.

[0036] Example 2 Analysis of the in vitro antibacterial activity of antimicrobial peptide PR-7 against Escherichia coli.

[0037] This embodiment systematically evaluates the in vitro antibacterial activity and antibacterial effect of the antimicrobial peptide PR-7 against Escherichia coli through experiments such as minimum inhibitory concentration (MIC) determination, time-kill curve plotting, and plate pouring method verification.

[0038] (1) Determination of minimum inhibitory concentration.

[0039] Test strains: indicator bacteria E. coli (Escherichia coli NBRC 3301, from the Japan NBRC Culture Collection Center); Preparation of bacterial suspension: Indicator bacteria E. coli Activated and passaged to the third generation using LB liquid medium, the third generation indicator bacteria were cultured at 37°C with shaking for 18 hours to reach the logarithmic growth phase. The bacteria were then transferred to sterile phosphate-buffered saline (PBS). E. coli The concentration was adjusted to 1×10 6 CFU / mL.

[0040] Grouping: Dissolve 5 mg of the antimicrobial peptide PR-7 sample in 500 μL PBS, sterilize by filtering through a 0.22 μM filter membrane, and then mix with an equal volume of 1×10⁻⁶ PBS. 6CFU / mL bacterial suspension was mixed to achieve final concentrations of antimicrobial peptide PR-7 of 1024 μM, 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, and 4 μM; a blank control group was set up (sterile PBS mixed with 10 μL of bacterial suspension).

[0041] After incubating at 37℃ for 8 hours to allow the antimicrobial peptide PR-7 to fully exert its effect, 100 μL of the reaction solution was spread and incubated in a 37℃ incubator for 18 hours.

[0042] Antimicrobial peptide PR-7 E. coli The antibacterial activity results showed that when PR-7 was effective against... E. coli The minimum inhibitory concentration (MIC) is 64 μM.

[0043] (2) Determination of antibacterial rate and plotting of time-kill curve: Cultured using the same method as above E. coli Indicator bacteria. Indicator bacteria E. coli The suspension was mixed with antimicrobial peptide PR-7 solution or PBS in 96-well plates and incubated to achieve final concentrations of antimicrobial peptide PR-7 of 1024 μM, 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, and 4 μM. PBS served as a blank control. Samples were taken at 0h, 2h, 4h, 6h, and 8h, and the OD of each well was measured using a microplate reader. 600 The antibacterial rate is calculated using the following formula: Growth inhibition rate (%) = (0D0j - (0Dij - 0Di0) / 0D0j) × 100%.

[0044] In the formula, OD0j represents 10 μL of indicator bacteria. E. coli The absorbance of the suspension and 50 μL of sterile water, with ODii as the indicator bacteria in 10 μL. E. coli The absorbance of the suspension and 50 μL of antimicrobial peptide PR-7 solution, ODi0 is the absorbance of 10 μL of sterile water and 50 μL of antimicrobial peptide PR-7 solution.

[0045] Multiple candidate peptides (PR-2, PR-3, PR-5, PR-6, PR-7) at concentrations of 1024 μM, 256 μM, 128 μM, 64 μM, 32 μM, 16 μM, 8 μM, and 4 μM were mixed with indicator bacteria. E. coli The suspension was mixed and incubated at 37℃ for 8 hours. The absorbance was measured, and the inhibition rate was calculated. The results are as follows: Figure 1 As shown, under the same test conditions (1024 μM at 8 h), the antimicrobial peptide PR-7 showed a significantly higher inhibition rate against Escherichia coli than other candidate peptides, exhibiting the best antimicrobial effect.

[0046] Calculate the antibacterial rate for each gradient, such as Figure 2As shown. Results PR-7 for E. coli It exhibits concentration-dependent antibacterial activity, with the antibacterial rate significantly increasing as the concentration of the antimicrobial peptide increases; it can inhibit Escherichia coli at a concentration of 64 μM.

[0047] Using the plate pouring method, 100 μL of the solutions obtained after incubating the above-mentioned gradient concentrations of PR-7 with bacterial suspension for 8 hours were poured into sterile petri dishes, then poured into sterilized LB solid medium cooled to 60℃, and shaken in a figure-eight motion 3-4 times. The dishes were then incubated at 37℃ for 24 hours to observe the antibacterial effect. Results are as follows... Figure 3 As shown, when the PR-7 concentration is ≥64 μM, no Escherichia coli colonies grow on the plate; when the concentration is below 64 μM, colony growth of varying degrees can be observed, further visually verifying that the minimum inhibitory concentration of PR-7 against Escherichia coli is 64 μM.

[0048] In summary, this invention addresses the core industry pain points of the current crisis of bacterial resistance caused by the overuse of traditional antibiotics, the frequent safety controversies surrounding chemical preservatives in the food industry, and the general shortcomings of existing antimicrobial peptides, such as insufficient antibacterial activity, poor biosafety, easy induction of drug resistance, and limited industrial application. It provides a cationic antimicrobial peptide, PR-7. Through systematic bioinformatics prediction, multi-dimensional in vitro activity verification, drug resistance evaluation, and biosafety analysis, this invention comprehensively demonstrates that the antimicrobial peptide possesses excellent overall performance, filling a gap in existing technologies and providing a novel core candidate molecule for the development of new antimicrobial products. It also possesses significant theoretical innovation value and broad prospects for industrial application.

[0049] The antimicrobial peptide PR-7 provided by this invention, with its amino acid sequence shown in SEQ ID NO.1, has been verified by a multi-platform bioinformatics system to possess the typical core characteristics of a highly efficient cationic antimicrobial peptide: strong basicity, suitable molecular weight, a net positive charge of +3, and a suitable hydrophilic-hydrophobic balance. Simultaneously, its Boman index is within the optimal range for drug development, exhibiting low protein binding potential and excellent expected bioavailability in vivo. This molecular design ensures its antimicrobial activity and application potential, exerting its effect by disrupting the surface structure of bacteria. PR-7 is less prone to inducing drug resistance and possesses high cell selectivity, making it suitable for preparing antimicrobial compositions for treating Escherichia coli infections. Furthermore, its minimum inhibitory concentration (MIC) is 64 μM, demonstrating significantly superior antimicrobial efficacy compared to other candidate peptides under the same conditions. The antimicrobial activity exhibits a clear concentration- and time-dependent effect, achieving complete inhibition of the target pathogen within an 8-hour treatment period, demonstrating rapid and efficient antimicrobial capability.

[0050] Therefore, the antimicrobial peptide PR-7 of this invention can not only be used as a core active ingredient to prepare antimicrobial drugs and antimicrobial compositions for the prevention and treatment of Escherichia coli infections, providing a novel solution for the treatment of multidrug-resistant bacterial infections in clinical practice; it can also be used as a natural antimicrobial additive in the field of food preservation, replacing traditional chemical preservatives. While inhibiting the growth of pathogenic bacteria in food and extending shelf life, it avoids the health risks of chemical additives, meeting the current core demand of consumers for natural and safe food.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. An antimicrobial peptide PR-7, characterized in that, The amino acid sequence of the antimicrobial peptide PR-7 is shown in SEQ ID NO.

1.

2. The use of the antimicrobial peptide PR-7 according to claim 1 in the preparation of antimicrobial agents.

3. The application according to claim 2, characterized in that, Antibacterial agents are used to inhibit Escherichia coli.

4. The application according to claim 3, characterized in that, The minimum inhibitory concentration of the antimicrobial peptide PR-7 against Escherichia coli is 64 μM.

5. The use of the antimicrobial peptide PR-7 according to claim 1 in the preparation of a drug against Escherichia coli.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition is a pharmaceutical composition with the antimicrobial peptide PR-7 as the main component according to claim 1.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is an injection, oral preparation, topical preparation, spray, or lyophilized preparation.

8. An antibacterial additive, characterized in that, The antimicrobial additive includes the antimicrobial peptide PR-7 according to claim 1, and the antimicrobial additive is added to food as an additive for use.

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